Combretastatin analogs with tubulin binding activity

ABSTRACT

Analogs of combretastatin have been discovered which demonstrate impressive cytotoxicity as well as a remarkable ability to inhibit tubulin polymerization. Such compounds are excellent clinical candidates for the treatment of cancer in humans. In addition, certain of these ligands, as pro-drugs, may well prove to be tumor selective vascular targeting chemotherapeutic agents or to have vascular targeting activity resulting in the selective prevention and/or destruction of nonmalignant proliferating vasculature.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/690,689, filed Jun. 14, 2005, entitled “Combretastatin Analogs withTubulin Binding Activity”. The entire content of the above-referencedapplication is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The cytoskeletal protein tubulin is among the most attractivetherapeutic drug targets for the treatment of solid tumors. Aparticularly successful class of chemotherapeutics mediates itsanti-tumor effect through a direct binding interaction with tubulin.This clinically promising class of therapeutics, called tubulin bindingagents or anti-tubulin agents, exhibit potent tumor cell cytotoxicity byefficiently inhibiting the assembly of α,β-tubulin heterodimers intomicrotubule structures that are required to facilitate mitotic celldivision (Li & Sham, Expert Opin. Ther. Patents., 2002).

Currently, the most widely recognized and clinically useful anti-tubulinchemotherapeutics agents are the Vinca Alkaloids, such as Vinblastineand Vincristine (Owellen et al., Cancer Res., 1976) along with Taxanessuch as Taxol (Schiff et al., Nature, 1979). Additionally, naturalproducts such as Rhizoxin (Rao et al., Tetrahedron Lett., 1992), theCombretastatins (Pettit et al., Can. J. Chem., 1982), Curacin A (Gerwicket al., J. Org. Chem., 1994), Podophyllotoxin (Coretese et al., I. Biol.Chem., 1977), Epothilones A and B (Nicolau et al., Nature, 1997),Dolastatin-10 (Pettit et al., J. Am. Chem. Soc, 1987), and Welwistatin(Zhang et al., Molecular Pharmacology, 1996), as well as certainsynthetic analogs including Phenstatin (Pettit G R et al., J. Med.Chem., 1998), 2-styrylquinazolin-4(3H)-ones (“SQOs”, Jiang et al., J.Med. Chem., 1990), highly oxygenated derivatives of cis- andtrans-stilbene, and dihydrostilbene (Cushman et al., J. Med. Chem.,1991) are all known to mediate tumor cytotoxic activity through a modeof action that includes tubulin binding and subsequent inhibition ofmitosis.

Normally, during the metaphase of cell mitosis, the nuclear membrane hasbroken down and tubulin is able to form centrosomes (also calledmicrotubule organizing centers) that facilitate the formation of themicrotubule spindle apparatus to which the dividing chromosomes becomeattached. Subsequent assembly and disassembly of the spindle apparatusmitigates the separation of the daughter chromosomes during anaphasesuch that each daughter cell contains a full complement of chromosomes.As antiproliferatives or antimitotic agents, tubulin binding agentsexploit the relatively rapid mitosis that occurs in proliferating tumorcells. By binding to tubulin and inhibiting the formation of the spindleapparatus in a tumor cell, the tubulin binding agent can causesignificant tumor cell cytotoxicity with relatively minor effects on theslowly dividing normal cells of the patient.

The exact nature of tubulin binding site interactions remains largelyunknown, and they definitely vary between each class of tubulin bindingagent. Photoaffinity labeling and other binding site elucidationtechniques have identified three key binding sites on tubulin: 1) theColchicine site (Williams et al., J. Biol. Chem., 1985); 2) the VincaAlkaloid site (Safa et al., Biochemistry, 1987); and 3) a site on thepolymerized microtubule to which taxol binds (Lin et al., Biochemistry,1989). An important aspect of this work requires a detailedunderstanding, at the molecular level, of the “small molecule” bindingdomain of both the α and β subunits of tubulin. The tertiary structureof the α,β tubulin heterodimer was reported in 1998 by Downing andco-workers at a resolution of 3.7 Å using a technique known as electroncrystallography (Nogales et al., Nature, 1998). This brilliantaccomplishment culminated decades of work directed toward theelucidation of this structure and should facilitate the identificationof small molecule binding sites, such as the colchicine site, usingtechniques such as photoaffinity and chemical affinity labeling (Chavanet al., Bioconjugate Chem., 1993; Hahn et al., Photochem. Photobio L,1992).

Further significance is given to new drugs that bind to the colchicinesite since it has recently been shown that many tubulin binding agentsalso demonstrate activity against malignant proliferating tumorvasculature, as opposed to the tumor itself. Antivascular chemotherapyis an emerging area of cancer chemotherapy which centers on thedevelopment of drugs that target the proliferation of the vasculaturethat supports tumor growth. Much of the research in anti-vascular cancertherapy has focused on understanding the process of new blood vesselformation, known as angiogenesis, and identifying anti-angiogenic agentswhich inhibit the formation of new blood vessels. Angiogenesis ischaracterized by the proliferation of tumor endothelial cells andgeneration of new vasculature to support the growth of a tumor. Thisgrowth is stimulated by certain growth factors produced by the tumoritself. One of these growth factors, Vascular Endothelial Growth Factor(“VEGF”), is relatively specific towards endothelial cells, by virtue ofthe restricted and up-regulated expression of its cognate receptor.Various anti-angiogenic strategies have been developed to inhibit thissignaling process at one or more steps in the biochemical pathway inorder to prevent the growth and establishment of the tumor vasculature.However, anti-angiogenic therapies act slowly and must be chronicallyadministered over a period of months to years in order to produce thedesired effect.

Vascular Targeting Agents (“VTAs”), also known as vascular disruptingagents or vascular damaging agents, are a separate class of antivascularchemotherapeutics. In contrast to anti-angiogenic drugs which disruptthe new microvessel formation of developing tumors, VTAs attack solidtumors by selectively targeting the established tumor vasculature andcausing extensive shutdown of tumor blood flow. A single dose of a VTAcan cause a rapid and selective shutdown of the tumor neovasculaturewithin a period of minutes to hours, leading eventually to tumornecrosis by induction of hypoxia and nutrient depletion. Thisvascular-mediated cytotoxic mechanism of VTA action is quite divorcedfrom that of anti-angiogenic agents, which inhibit the formation of newtumor vascularization rather than interfering with the existing tumorvasculature. Other agents have been known to disrupt tumor vasculature,but differ in that they also manifest substantial normal tissue toxicityat their maximum tolerated dose. In contrast, genuine VTAs retain theirvascular shutdown activity at a fraction of their maximum tolerateddose. It is thought that tubulin-binding VTAs selectively destabilizethe microtubule cytoskeleton of tumor endothelial cells, causing aprofound alteration in the shape of the cell which ultimately leads toocclusion of the tumor blood vessel and shutdown of blood flow to thetumor (Kanthou et al., Blood, 2002).

Combretastatin A4 phosphate prodrug (CA4P) is one of the leading newcandidates from among a relatively small collection of known worldcompounds with vascular targeting activity (U.S. Pat. No. 5,561,122;Chaplin et al., Anticancer Res., 1999; Tozer et al., Cancer Res., 1999;Pettit and Rhodes, Anti-Cancer Drug Des., 1998; Iyer et al., CancerRes., 1998; Dark et al., Cancer Res., 1997). Its parent phenol compound,Combretastatin A-4 (CA4) was discovered by Professor George R. Pettit(Arizona State University) as an isolate from South African bush willow(Combretum caffrum) in the 1970s. CA4 is a potent inhibitor of tubulinpolymerization and binds to the colchicine site on β-tubulin.Interestingly, CA4 itself does not demonstrate destruction of tumorvasculature, while CA4P is very active in terms of tumor vasculaturedestruction. Therefore, the phosphate ester portion of CA4P undergoesdephosphorylation to reveal the potent tubulin binder CA4 that destroysthe tumor cell through an inhibition of tubulin polymerization.

CA4P is currently the lead drug in a group of tubulin-binding VTAs underclinical development. Other tubulin binding VTAs that have beendiscovered include the colchicinoid ZD6126 (Davis et al., CancerResearch, 2002) and the Combretastatin analog AVE8032 (Lejeune et al.,Proceedings of the AACR., 2002). Despite these advances, an aggressivechemotherapeutic strategy for the treatment and maintenance of solidtumor cancers continues to rely on the development of architecturallynew and biologically more potent compounds. The present inventionaddresses this urgent need by providing a structurally novel class oftubulin binding agent compositions with potent antiproliferativeactivity and tumor cell cytotoxicity.

SUMMARY OF THE INVENTION

The present invention relates to a discovery of Combrestatin analogswhich function as tubulin binding agents capable of inhibiting tubulinassembly and tumor cell proliferation. These Combrestatin analogs resultfrom the judicious combination of a non-tubulin binding moleculartemplate, suitably modified with structural features such as hydroxylmoieties and arylalkoxy groups. Furthermore, the invention providescompounds useful in the treatment, prevention or amelioration of one ormore symptoms of vascular proliferative disorders and neoplasticdiseases. The compounds of the invention are also useful in reducing theflow of blood to at least a portion of a neoplastic region, and forinhibiting tubulin polymerization.

In one general aspect, the present invention provides a Combretastatinanalog of the following general formula I:

or a pharmaceutically acceptable salt thereof, wherein

the dashed lines indicate a single or double bond;

R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each, independently, selected from thegroup consisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl,amine, phosphate, phosphoramidate, and amino acid acyl group;

X is selected from the group consisting of a single bond, CH₂, O, S,N(H), and C(O); and n is 0, 1, 2 or 3.

In one embodiment of Formula I, R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each,independently, selected from the group consisting of H, lower alkoxy,hydroxyl, phosphate and phosphoramidate. In another embodiment ofFormula I, X is a single bond. In another embodiment of Formula I, n is2 or 3.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula II:

or a pharmaceutically acceptable salt thereof,

wherein the dashed lines indicate a single or double bond;

R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each, independently, selected from thegroup consisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl,amine, phosphate, phosphoramidate, and amino acid acyl group;

X is selected from the group consisting of a single bond, CH₂, O, S,N(H), and

C(O); and n is 0, 1, 2 or 3.

In another embodiment of Formula II, R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, loweralkoxy, hydroxyl, phosphate and phosphoramidate. In yet anotherembodiment of Formula II, X is a single bond. In another embodiment ofFormula II, R₁, R₂ and R₃, are each, independently, selected from thegroup consisting of H, OCH₃, phosphate and OH. In still anotherembodiment of Formula II, R₄, R₅ and R₆, are each, independently,selected from the group consisting of H, OCH₃, phosphate and OH. Instill another embodiment of Formula II, R₇ is H. In still anotherembodiment of Formula II, n is 2 or 3.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IIa:

or a pharmaceutically acceptable salt thereof,

wherein the phenyl ring “Z” is bonded to either carbon “a” or “b”;

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, hydroxyl, and phosphate;

X is selected from the group consisting of a single bond and C(O); and

n is 1, 2, 3 or 4.

In one embodiment of formula IIa, R₄ and R₅ are OCH₃. In anotherembodiment of Formula IIa, n is 1. In another embodiment of Formula IIa,n is 3. In another embodiment of Formula IIa, n is 4.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IIb:

or a pharmaceutically acceptable salt thereof,

wherein the phenyl ring “Z” is bonded to either carbon “a” or “b”;

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, hydroxyl, and phosphate;

X is selected from the group consisting of a single bond and C(O); and nis 1, 2, 3 or 4.

In one embodiment of formula IIb, R₄ is H or OH, and R₅ is OH.

In particular embodiments of formula IIa or IIb, R₁ and R₃ are H. Inother embodiments of formula IIa or IIb, R₁ is OH and R₃ is H. In yetother embodiments of formula IIa or IIb, R₁ and R₃ are OCH₃. In anotherembodiment of Formula IIb, n is 1. In another embodiment of Formula IIb,n is 3. In another embodiment of Formula IIb, n is 4.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula III:

or a pharmaceutically acceptable salt thereof, wherein:

the dashed line indicates a single or double bond;

X is selected from the group consisting of a single bond, CH₂, O, S,N(H), and C(O);

and R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each, independently, selected fromthe group consisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl,amine, phosphate, phosphoramidate, and amino acid acyl group.

In one embodiment of Formula III, X is a single bond, and R₁, R₂ and R₃,are each, independently, selected from the group consisting of H, OCH₃,phosphate and OH. In another embodiment of Formula III, X is a singlebond, and R₄, R₅ and R₆, are each, independently, selected from thegroup consisting of H, OCH₃, phosphate and OH.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula IV:

or a pharmaceutically acceptable salt thereof, wherein:

the dashed lines independently indicate a single or double bond;

X is selected from the group consisting of a single bond, CH₂, O, S,N(H), and C(O);

R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each, independently, selected from thegroup consisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl,amine, phosphate, phosphoramidate, and amino acid acyl group;

and phenyl ring “Z” is bonded to either carbon “a” or “b.”

In another embodiment of Formula IV, the dashed lines are single bonds,X is a single bond, and R₁, R₂ and R₃, are each, independently, selectedfrom the group consisting of H, OCH₃, phosphate and OH. In still anotherembodiment of Formula IV, the dashed lines are single bonds, X is asingle bond, and R₄, R₅ and R₆, are each, independently, selected fromthe group consisting of H, OCH₃, phosphate and OH. In still anotherembodiment of Formula IV, the dashed lines are single bonds, X is asingle bond, and the phenyl ring “Z” is bonded to carbon “a.” In yetanother embodiment of Formula IV, the dashed lines are single bonds, Xis a single bond, and phenyl ring “Z” is bonded to carbon “b.”

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IVa:

or a pharmaceutically acceptable salt thereof,wherein the phenyl ring “Z” is bonded to either carbon “a” or “b”; and

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, hydroxyl, and phosphate.

In one embodiment of formula IVa, R₁ and R₃ are OCH₃. In anotherembodiment of formula IVa, R₄ and R₅ are OH. In another embodiment offormula IVa, R₄ and R₅ are phosphate. In yet another embodiment offormula IVa, phenyl ring “Z” is bonded to carbon “a.”

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IVb:

or a pharmaceutically acceptable salt thereof, wherein the phenyl ring“Z” is bonded to either carbon “a” or “b”; and

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, hydroxyl, and phosphate.

In one embodiment of formula IVb, R₁ and R₃ are OCH₃. In anotherembodiment of formula IVb, R₄ and R₅ are OH. In another embodiment offormula IVb, R₄ and R₅ are phosphate. In yet another embodiment offormula IVb, phenyl ring “Z” is bonded to carbon “a.”

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula V:

or a pharmaceutically acceptable salt thereof, wherein R₆ is selectedfrom the group consisting of H, halogen, lower alkyl, lower alkoxy,hydroxyl, amine, phosphate, phosphoramidate, and amino acid acyl group.

In another embodiment of Formula V, R₆ is selected from the groupconsisting of H, OCH₃, phosphate and OH. In yet another embodiment ofFormula V, R₆ is OH.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula VI:

or a pharmaceutically acceptable salt thereof, wherein:

R₁, R₃, R₅ and R₆ are each, independently, selected from the groupconsisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl, amine,phosphate, phosphoramidate, and amino acid acyl group;

and phenyl ring “Z” is bonded to either carbon “a” or “b.”

In one embodiment of Formula VI, R₁ and R₃ are selected from the groupconsisting of H, OCH₃, phosphate and OH. In another embodiment ofFormula VI, R₅ and R₆, are each, independently, selected from the groupconsisting of H, OCH₃, phosphate and OH. In still another embodiment ofFormula VI, the phenyl ring “Z” is bonded to carbon “a.” In anotherembodiment of Formula VI, the phenyl ring “Z” is bonded to carbon “b.”

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula VII:

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R₄,R₅, R₆, and R₇ are each, independently, selected from the groupconsisting of OH, phosphate, and OCH₃.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula VIII:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindicate a single or double bond; R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, halogen,lower alkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate,and amino acid acyl group; X is selected from the group consisting of asingle bond, CH₂, O, S, N(H), and C(O); X₁, X₂, X₃, X₄ and X₅ are each,independently, selected from the group consisting of C, C(H), N, N(H), Oand S, provided that at least one of X₁, X₂, X₃, X₄ and X₅ is not C orC(H); and n is 0, 1, 2 or 3.

In one embodiment of Formula VIII, R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, loweralkoxy, hydroxyl, phosphate and phosphoramidate. In another embodimentof Formula VIII, X is a single bond. In another embodiment of FormulaVIII, n is 0. In another embodiment of Formula VIII, n is 2. In anotherembodiment of Formula VIII, n is 3.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula IX:

or a pharmaceutically acceptable salt thereof, wherein the dashed lineindicates a single or double bond; X is selected from the groupconsisting of a single bond, CH₂, O, S, N(H), and C(O); X₁, X₂, X₃, X₄and X₅ are each, independently, selected from the group consisting of C,C(H), N, N(H), O and S, provided that at least one of X₁, X₂, X₃, X₄ andX₅ is not C or C(H); R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each,independently, selected from the group consisting of H, halogen, loweralkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate, andamino acid acyl group, and ring “Z” is bonded to either carbon “a” or“b.” In one embodiment of Formula IX, X is a single bond, and R₁, R₂ andR₃, are each, independently, selected from the group consisting of H,OCH₃, phosphate and OH. In another embodiment of Formula IX, X is asingle bond, and R₄, R₅ and R₆, are each, independently, selected fromthe group consisting of H, OCH₃, phosphate and OH.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula X:

or a pharmaceutically acceptable salt thereof, wherein R₁, R₃, R₅ and R₆are each, independently, selected from the group consisting of H,halogen, lower alkyl, lower alkoxy, hydroxyl, amine, phosphate,phosphoramidate, and amino acid acyl group, X₁, X₂, X₃, X₄ and X₅ areeach, independently, selected from the group consisting of C, C(H) andN, provided that at least one of X₁, X₂, X₃, X₄ and X₅ is not C or C(H);and phenyl ring “Z” is bonded to either carbon “a” or “b.” In one aspectof Formula X, R₁ and R₃ are selected from the group consisting of H,OCH₃, phosphate and OH. In another aspect of Formula X, R₅ and R₆, areeach, independently, selected from the group consisting of H, OCH₃,phosphate and OH. In still another aspect of Formula X, the phenyl ring“Z” is bonded to carbon “a.” In another aspect of Formula X, the phenylring “Z” is bonded to carbon “b.”

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula XII:

or a pharmaceutically acceptable salt thereof, wherein R₁, R₄, R₅ and R₆are each, independently, selected from the group consisting of OH andOCH₃ and X₁, X₂, X₃, X₄ and X₅ are each, independently, selected fromthe group consisting of C, C(H) and N, provided that at least one of X₁,X₂, X₃, X₄ and X₅ is not C or C(H).

In another general aspect, the invention provides a method for treatinga vascular proliferative disorder in an animal comprising administeringto an animal an effective amount of a ring-substituted bicyclic fusedring system, wherein the centroid to centroid distance between the ringsubstituent and the outer ring of the bicyclic fused ring system isbetween 4 and 7 Å. In one embodiment, the centroid to centroid distancebetween the ring substituent and the outer ring of the bicyclic fusedring system is between 4 and 5 Å. In another embodiment, the centroid tocentroid distance between the ring substituent and the outer ring of thebicyclic fused ring system is between 5 and 6 Å. In still anotherembodiment, the centroid to centroid distance between the ringsubstituent and the outer ring of the bicyclic fused ring system isbetween 6 and 7 Å. In another embodiment, the ring-substituted bicyclicfused ring system is represented by a compound of the Formula I, II,IIa, IIb, III, IV, IVa, IVb, V, VI, VII, VIII, IX, X and XII. In anotherembodiment, the ring-substituted bicyclic fused ring system is selectedfrom the group consisting of3-Methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene;3-Methoxy-9-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-phenol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-benzene-1,2-diol;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanoneand(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanone.

In another aspect, the invention provides a method for selectivelyreducing the flow of blood to at least a portion of a neoplastic region,comprising administering a ring-substituted bicyclic fused ring system,wherein the centroid to centroid distance between the ring substituentand the outer ring of the bicyclic fused ring system is between 4 and 7Å, thereby causing substantial necrosis of tissue in the neoplasticregion without substantial necrosis of tissue in adjoining regions. Inone embodiment, the centroid to centroid distance between the ringsubstituent and the outer ring of the bicyclic fused ring system isbetween 4 and 5 Å. In another embodiment, the centroid to centroiddistance between the ring substituent and the outer ring of the bicyclicfused ring system is between 5 and 6 Å. In still another embodiment, thecentroid to centroid distance between the ring substituent and the outerring of the bicyclic fused ring system is between 6 and 7 Å. In anotherembodiment, the ring-substituted bicyclic fused ring system isrepresented by a compound of the Formula I, II, IIa, IIb, III, IV, IVa,IVb, V, VI, VII, VIII, IX, X and XII. In another embodiment, thering-substituted bicyclic fused ring system is selected from the groupconsisting of3-Methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene;3-Methoxy-9-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H -benzocycloheptene;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-phenol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-benzene-1,2-diol;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanoneand(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanone.

In another embodiment, the ring-substituted bicyclic fused ring systemis selected from the group consisting of3-Methoxy-6-(4,5,6-trimethoxy-3H-inden-1-yl)-benzene-1,2-diol;2-Methoxy-5-(4,5,6-trimethoxy-3H-inden-1-yl)-phenol;2-Methoxy-5-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-phenol;2-Methoxy-5-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-1-yl)-phenol;2-Methoxy-5-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-2-yl)-phenol;3-Methoxy-6-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-1-yl)-benzene-1,2-diol;3-Methoxy-6-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-2-yl)-benzene-1,2-diol;(2,3-Dihydroxy-4-methoxy-phenyl)-(4,5,6-trimethoxy-3H-inden-1-yl)-methanone;(3-Hydroxy-4-methoxy-phenyl)-(4,5,6-trimethoxy-3H-inden-1-yl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanoneand(3-Hydroxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanone.

In another embodiment, the reduction in tumor blood flow is reversiblesuch that normal tumor blood flow is restored following cessation oftreatment.

In another aspect, the invention provides a method for treatingneoplastic disease in an animal comprising administering to an animal aring-substituted bicyclic fused ring system, wherein the centroid tocentroid distance between the ring substituent and the outer ring of thebicyclic fused ring system is between 4 and 7 Å. In one embodiment, thecentroid to centroid distance between the ring substituent and the outerring of the bicyclic fused ring system is between 4 and 5 Å. In anotherembodiment, the centroid to centroid distance between the ringsubstituent and the outer ring of the bicyclic fused ring system isbetween 5 and 6 Å. In still another embodiment, the centroid to centroiddistance between the ring substituent and the outer ring of the bicyclicfused ring system is between 6 and 7 Å. In another embodiment, thering-substituted bicyclic fused ring system is represented by a compoundof the Formula I, II, IIa, IIb, III, IV, IVa, IVb, V, VI, VII, VIII, IX,X and XII. In another embodiment, the ring-substituted bicyclic fusedring system is selected from the group consisting of3-Methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene;3-Methoxy-9-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-phenol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-benzene-1,2-diol;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanoneand(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanone.

In another embodiment, the ring-substituted bicyclic fused ring systemis selected from the group consisting of3-Methoxy-6-(4,5,6-trimethoxy-3H-inden-1-yl)-benzene-1,2-diol;2-Methoxy-5-(4,5,6-trimethoxy-3H-inden-1-yl)-phenol;2-Methoxy-5-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-phenol;2-Methoxy-5-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-1-yl)-phenol;2-Methoxy-5-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-2-yl)-phenol;3-Methoxy-6-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-1-yl)-benzene-1,2-diol;3-Methoxy-6-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-2-yl)-benzene-1,2-diol;(2,3-Dihydroxy-4-methoxy-phenyl)-(4,5,6-trimethoxy-3H-inden-1-yl)-methanone;(3-Hydroxy-4-methoxy-phenyl)-(4,5,6-trimethoxy-3H-inden-1-yl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanoneand(3-Hydroxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanone.

In another embodiment, the compound has the direct result of causingtumor cell cytotoxicity due to inhibition of mitosis.

In another aspect, the invention provides a method for inhibitingtubulin polymerization by contacting a tubulin-containing system with aring-substituted bicyclic fused ring system, wherein the centroid tocentroid distance between the ring substituent and the outer ring of thebicyclic fused ring system is between 4 and 7 Å. In one embodiment, thecentroid to centroid distance between the ring substituent and the outerring of the bicyclic fused ring system is between 4 and 5 Å. In anotherembodiment, the centroid to centroid distance between the ringsubstituent and the outer ring of the bicyclic fused ring system isbetween 5 and 6 Å. In still another embodiment, the centroid to centroiddistance between the ring substituent and the outer ring of the bicyclicfused ring system is between 6 and 7 Å. In another embodiment, thering-substituted bicyclic fused ring system is represented by a compoundof the Formula I, II, IIa, IIb, III, IV, IVa, IVb, V, VI, VII, VIII, IX,X and XII. In another embodiment, the ring-substituted bicyclic fusedring system is selected from the group consisting of3-Methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene;3-Methoxy-9-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol;2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-7,8,9,10-tetrahydro-benzocycloocten-1-ol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-benzene-1,2-diol;2-Methoxy-5-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-phenol;2-Methoxy-5-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-phenol;3-Methoxy-6-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-benzene-1,2-diol;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanoneand(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-7,8,9,10-tetrahydro-benzocycloocten-5-yl)-methanone.

In another embodiment, the ring-substituted bicyclic fused ring systemis selected from the group consisting of3-Methoxy-6-(4,5,6-trimethoxy-3H-inden-1-yl)-benzene-1,2-diol;2-Methoxy-5-(4,5,6-trimethoxy-3H-inden-1-yl)-phenol;2-Methoxy-5-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-phenol;2-Methoxy-5-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-1-yl)-phenol;2-Methoxy-5-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-2-yl)-phenol;3-Methoxy-6-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-1-yl)-benzene-1,2-diol;3-Methoxy-6-(6,7,8-trimethoxy-3,4-dihydro-naphthalen-2-yl)-benzene-1,2-diol;(2,3-Dihydroxy-4-methoxy-phenyl)-(4,5,6-trimethoxy-3H-inden-1-yl)-methanone;(3-Hydroxy-4-methoxy-phenyl)-(4,5,6-trimethoxy-3H-inden-1-yl)-methanone;(2,3-Dihydroxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanoneand(3-Hydroxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanone.

In another embodiment, the system is a tumor cell.

In another aspect, the invention provides a pharmaceutical formulationcontaining a compound of Formula I, II, IIa, IIb, III, IV, IVa, IVb, V,VI, VII, VIII, IX, X and XII in a pharmaceutically suitable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a route for the synthesis of Compounds 1 and 2, exemplarycompounds of the invention.

FIG. 2 depicts a route for the synthesis of Compounds 3 and 4, exemplarycompounds of the invention.

FIG. 3 depicts a route for the synthesis of Compounds 5 and 6, exemplarycompounds of the invention.

FIG. 4 depicts a route for the synthesis of various intermediates usedin the synthesis of the compounds of the invention, including a listingof literature references describing the synthesis of intermediates I,II, III and IV.

FIG. 5 depicts a route for the synthesis of Compounds 7, 8, 9, 10, 11,12, 13 and 14, exemplary compounds of the invention.

FIG. 6 depicts a route for the synthesis of Compounds 15, 16, 17 and 18,exemplary compounds of the invention.

FIG. 7 depicts a route for the synthesis of Compounds 19 and 20,exemplary compounds of the invention.

FIG. 8 depicts a route for the synthesis of Compounds 21 and 22,exemplary compounds of the invention.

FIG. 9 depicts a route for the synthesis of Compounds 23 and 24,exemplary compounds of the invention.

FIG. 10 depicts a route for the synthesis of Compounds 25 and 26,exemplary compounds of the invention.

FIG. 11 depicts a route for the synthesis of intermediate I, anintermediate used in the preparation of some of compounds of theinvention.

FIG. 12 depicts a route for the synthesis of intermediate II, anintermediate used in the preparation of some of compounds of theinvention.

FIG. 13 depicts a route for the synthesis of an exemplary phosphateprodrug of the invention and pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The discovery of new anti-mitotic agents has resulted from the judiciouscombination of a molecular template (scaffold) that interacts withestrogen receptor (ER), and is modified with structural features deemedimperative for tubulin binding (i.e. hydroxyl, arylalkoxy groups,certain halogen substitutions, etc.). In particular, the methoxy arylfunctionality seems important for increased interaction at thecolchicine binding site in certain analogs (Shirai et al., BiomedicalChem. Lett. 1994). Our initial design and synthesis efforts centered onbenzo[b]thiophene ligands containing structural motifs reminiscent ofraloxifene, the selective estrogen receptor modulator (SERM) developedby Eli Lilly and Co. (Jones et al., J. Med Chem. 1984; Grese et al., J.Med Chem., 1997; Palkowitz et al., J. Med Chem., 1997), as well thecolchicine and combretastatin tubulin binding agents.

The design premise that molecular skeletons of traditional estrogenreceptor (ER) binding compounds can be modified with structural motifsreminiscent of colchicine and combretastatin A4 to produce especiallyinhibitors of tubulin polymerization has been validated by ourpreparation of very active benzo[b]thiophene, benzo[b]furan, and indoleantitubulin and anti-mitotic agents (U.S. Pat. Nos. 5,886,025;6,162,930; 6,350,777; and 6,593,374; PCT publication no. WO 01/19794;Mullica et al., J. Chem. Cryst., 1998; Pinney, et al., Bioorg. Med.Chem. Lett., 1999). The lead compounds in each series demonstrateremarkable biological activity against a variety of human cancer celllines.

In further support of our hypothesis, recent studies have shown thatcertain estrogen receptor (ER) binding compounds (ex.2-methoxyestradiol) can interact with tubulin and inhibit tubulinassembly as structurally modified estradiol congeners (D'Amato et al.,Proc. Natl. Acad Sci., 1994; Cushman et al., J. Med Chem., 1995; Hamelet al., Biochemistry, 1996; Cushman et al., J. Med. Chem., 1997).Estradiol is perhaps the most important estrogen in humans, and it isintriguing and instructive that the addition of the methoxy aryl motifto this compound makes it interactive with tubulin. It is alsonoteworthy that 2-methoxyestradiol is a natural mammalian metabolite ofestradiol and may play a cell growth regulatory role especiallyprominent during pregnancy.

Our analysis of the structure-activity relationships ofbenzo[b]thiophene constructs has emphasized the importance of judiciousplacement of the trimethoxyphenyl ring and 4-methoxyphenyl rings.Without being bound by theory, pseudo pi stacking of the two aryl ringsalong with sp¹ hybridization at the bridge atoms between the rings isimportant for retaining tubulin binding properties. In addition to thesefactors, the centroid-to-centroid distances between the two-aryl ringsare important. Optimization of this distance to approach that of CA4(4.7 Å) improves the binding affinity of the molecule for thecolchicine-binding site of tubulin. Introduction of spacer moiety of twocontiguous atoms between the two rings improves tubulin bindingproperties by allowing the molecule more freedom to align itself forpseudo pi stacking. Similarly, restriction to free rotation by theintroduction of a double bond resulted in better biological activity.

Surprisingly, the novel Combrestatin analogs described herein retainantiproliferative and tubulin binding properties despite the presence a5, 6, 7, or 8-membered ring which confers increased flexibility to themolecule.

I) Definitions

As used herein, the following terms in quotations shall have theindicated meanings,

whether in plural or singular form:

“Amino acid acyl group” in the amino acid acylamino group is an acylgroup derived from the amino acid. The amino acids may be enumerated bya-amino acids, p-amino acids and y-amino acids. Examples of preferredamino acids include glycine, alanine, leucine, serine, lysine, glutamicacid, aspartic acid, threonine, valine, isoleucine, onithine, glutamine,asparagine, tyrosine, phenylalanine, cysteine, methionine, arginine,P-alanine, tryptophan, proline, histidine, etc. The preferred amino acidis serine and the preferred amino acid acyl group is a serinamide.

“Amine” refers to a free amine NH₂ or a lower alkylamino.

“Animal” refers to any warm-blooded mammal, preferably a human.

“Alkyl” refers to a group containing from 1 to 8 carbon atoms and may bestraight chained or branched. An alkyl group is an optionallysubstituted straight, branched or cyclic saturated hydrocarbon group.When substituted, alkyl groups may be substituted with up to foursubstituent groups, R as defined, at any available point of attachment.When the alkyl group is said to be substituted with an alkyl group, thisis used interchangeably with “branched alkyl group.” Exemplaryunsubstituted such groups include methyl, ethyl, propyl, isopropyl,n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl,dodecyl, and the like. Exemplary substituents may include but are notlimited to one or more of the following groups: halo (such as F, Cl, Br,I), haloalkyl (such as CCl3 or CF3), alkoxy, alkylthio, hydroxy, carboxy(—COOH), alkyloxycarbonyl (—C(O)R), alkylcarbonyloxy (—OCOR), amino(—NH2), carbamoyl (—NHCOOR— or —OCONHR—), urea (—NHCONHR—) or thiol(—SH). Alkyl groups as defined may also comprise one or more carbon tocarbon double bonds or one or more carbon to carbon triple bonds.

The term “aryl” herein includes 5- and 6-membered single-ring aromaticgroups that may include from zero to four heteroatoms, for example,benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like. Aryl groups also include polycyclic fusedaromatic groups such as naphthyl, quinolyl, indolyl, and the like. Thosearyl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles”, “heteroaryls” or “heteroaromatics”.The aromatic ring can be substituted at one or more ring positions withsuch substituents as described above, as for example, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, or an aromatic or heteroaromatic moiety. Aryl groups canalso be fused or bridged with alicyclic or heterocyclic rings which arenot aromatic so as to form a polycycle (e.g., tetralin).

The terms “alkenyl” and “alkynyl” include unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” meansan alkyl group, as defined above, but having from one to ten carbons,more preferably from one to six carbon atoms (e.g., “C₁-C₆-alkyl”) inits backbone structure. Likewise, “lower alkenyl,” “lower alkoxy” and“lower alkynyl” have similar chain lengths. Preferred alkyl groups arelower alkyls. Furthermore, the expression “C_(x)-C_(y)-alkyl”, wherein xis 1-5 and y is 2-10 indicates a particular alkyl group (straight- orbranched-chain) of a particular range of carbons. For example, theexpression C₁-C₄-alkyl includes, but is not limited to, methyl, ethyl,propyl, butyl, isopropyl, tert-butyl and isobutyl. Moreover, the termC₃₋₆-cycloalkyl includes, but is not limited to, cyclopropyl,cyclopentyl, and cyclohexyl. These alkyl groups, as well as cycloalkylgroups, may be further substituted.

The terms “heterocyclyl” or “heterocyclic group” include 3- to10-membered ring structures, more preferably 4- to 7-membered rings,which ring structures include one to four heteroatoms. Heterocyclylgroups include pyrrolidine, oxolane, thiolane, piperidine, piperazine,morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, and the like. The heterocyclic ring can besubstituted at one or more positions with such substituents as describedabove, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (includingalkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, or an aromatic orheteroaromatic moiety.

“Aroyl” refers to the —(C═O)-aryl groups, wherein aryl is defined ashereinabove. The aryl group is bonded to the core compound through acarbonyl bridge.

“Cycloalkyl” is a species of alkyl containing from 3 to 15 carbon atoms,without alternating or resonating double bonds between carbon atoms. Itmay contain from 1 to 4 rings. Exemplary unsubstituted such groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl,etc. Exemplary substituents include one or more of the following groups:halogen, alkyl, alkoxy, alkyl hydroxy, ammo, nitro, cyano, thiol and/oralkylthio.

“Halogen” or “Halo” refers to chlorine, bromine, fluorine or iodine.

“Lower alkoxy” refers to —O-alkyl groups, wherein alkyl is as definedhereinabove. The alkoxy group is bonded to the core compound through theoxygen bridge. The alkoxy group may be straight-chained or branched;although the straight-chain is preferred. Examples include methoxy,ethyloxy, propoxy, butyloxy, t-butyloxy, i-propoxy, and the like.Preferred alkoxy groups contain 1-4 carbon atoms, especially preferredalkoxy groups contain 1-3 carbon atoms. The most preferred alkoxy groupis methoxy.

“Lower alkylamino” refers to a group wherein one or two alkyl groups isbonded to an amino nitrogen, i.e., NH(alkyl). The nitrogen is the bridgeconnecting the alkyl group to the core compound. Examples include NHMe,NHEt, NHPr, and the like.

The term “heteroatom” includes an atom of any element other than carbonor hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “aryl aldehyde,” as used herein, includes compounds representedby the formula Ar—C(O)H, in which Ar is an aryl moiety (as describedabove) and —C(O)H is a formyl or aldehydo group

“Prodrug” refers to a precursor form of the drug which is metabolicallyconverted in vivo to produce the active drug. Preferred prodrugs of thepresent invention include the phosphate, phosphoramidate, or amino acidacyl groups as defined herein. The phosphate ester salt moiety may alsoinclude (—OP(O)(O-alkyl)₂ or (—OP(O)(O⁻NH₄ ⁺)₂). In preferredembodiments, a prodrug of the invention comprises a substitution of aphenolic moiety or amine moiety of the active drug with a phosphate,phosphoramidate, or amino acid acyl group. A wide variety of methods forthe preparation of prodrugs are known to those skilled in the art (see,for example, Pettit and Lippert, Anti-Cancer Drug Design, (2000), 15,203-216).

“Phenolic moiety” means herein a hydroxyl group when it refers to an Rgroup on an aryl ring.

“Phosphate”, “Phosphate moiety”, or “Phosphate prodrug salt” refers tophosphate disalt moiety (—OP(O)(O⁻M⁺)₂), a phosphate triester moiety(—OP(O)(OR)₂) or a phosphate ester salt moiety (—OP(O)(OR)(O⁻M⁺), whereM is a salt and R is chosen to be any appropriate alkyl or branchedalkyl substituent (the two R groups may be the same alkyl group or maybe mixed), or benzyl, or aryl groups. The salt M is advantageously Na, Kand Li, but the invention is not limited in this respect.

Phosphoramidate” refers to a phosphoramidate ester salt moiety(—NP(O)(OR)(O-M+), a phosphoramidate diester moiety (—NP(O)(OR)2), or aphosphoramidate disalt moiety (—NP(O)(O-M+)2), where M is a salt and Ris chosen to be any appropriate alkyl or branched alkyl substituent (thetwo R groups may be the same alkyl group or may be mixed), or benzyl, oraryl groups. The salt M is advantageously Na, K and Li, but theinvention is not limited in this respect.

“Salt” is a pharmaceutically acceptable salt and can include acidaddition salts such as the hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,acetates, benzoates, citrates, maleates, fumarates, succinates,lactates, and tartrates; alkali metal cations such as Na, K, Li; alkaliearth metal salts such as Mg or Ca; or organic amine salts such as thosedisclosed in PCT International Application Nos. WO02/22626 orWO00/48606, which are incorporated herein by reference in theirentireties. Exemplary organic amine salts are tromethamine (TRIS) saltsand amino acid salts (e.g. histidine salts) of the compounds of theinvention.

“Treating” (or “treat”) as used herein includes its generally acceptedmeaning which encompasses prohibiting, preventing, restraining, andslowing, stopping, or reversing progression, severity, of a resultantsymptom. As such, the methods of this invention encompass boththerapeutic and prophylactic administration.

“Tubulin Binding Agent” shall refer to a ligand of tubulin or a compoundcapable of binding to either Aβ-tubulin heterodimers or microtubules andinterfering with the assembly or disassembly of microtubules.

“Effective amount” refers to the amount or dose of the compound, uponsingle or multiple dose administration to the patient, which providesthe desired effect in the patient under diagnosis or treatment. Aneffective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances.

II) Compounds of the Invention

The compounds of the invention are Combretastatin analogs which arecharacterized by a bicyclic fused ring system substituted by a6-membered ring. Such compounds are referred to herein as a“ring-substituted bicyclic fused ring systems” or “Combretastatinanalogs.” For demonstration purposes, an example of a ring-substitutedbicyclic fused ring system is shown below:

The X and Y rings constitute the bicyclic fused ring component, and theZ ring constitutes the 6-membered ring component. The dashed linesindependently indicate a single or double line, and n can be 0, 1, 2 or3. The carbons of the ring-substituted bicyclic fused ring system shownbelow can be individually substituted with an additional functionalgroup, as is the case in Formula I, II, IIa, IIb, III, IV, IVa, IVb, V,VI, VII, VIII, IX, X and XII as demonstrated below. Examples of bicyclicfused ring systems include, but are not limited to,1,2-dihydro-naphthalene, 1,2,3,4-tetrahydro-naphthalene, naphthalene,6,7,8,9-tetrahydro-5H-benzocycloheptene,6,7-dihydro-5H-benzocycloheptene, 7H-benzocycloheptene,5,6,7,8,9,10-hexahydro-benzocyclooctene, 5H-benzocycloheptene,5,6,7,8-tetrahydro-benzocyclooctene, and 5,6-dihydro-benzocyclooctene,and 5,8-dihydro-benzocyclooctene. Examples of 6-membered rings include,but are not limited to, cyclohexane, cyclohexene, cyclohexa-1,3-dieneand benzene.

In a particular embodiment, the centroid to centroid distance betweenthe ring substituent (ring Z) and the outer ring of the bicyclic fusedring system (ring X) is between 4 and 7 Å. In a preferred embodiment,the centroid to centroid distance between the ring substituent and theouter ring of the bicyclic fused ring system is between 4 and 5 Å. Inanother embodiment, the centroid to centroid distance between the ringsubstituent and the outer ring of the bicyclic fused ring system isbetween 5 and 6 Å. In still another embodiment, the centroid to centroiddistance between the ring substituent and the outer ring of the bicyclicfused ring system is between 6 and 7 Å. As used herein, the term“centroid to centroid distance” refers to the distance between thecenter of geometries of the ring substituent of the ring-substitutedbicyclic fused ring system and the outer ring of the ring-substitutedbicyclic fused ring system. As used herein, the term “outer ring of thering-substituted bicyclic fused ring system” refers to the ring of thering-substituted bicyclic fused ring system that is not substituted by a6-membered ring.

In one general aspect, the present invention provides a Combretastatinanalog of the following general formula I:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindicate a single or double bond; R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, halogen,lower alkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate,and amino acid acyl group; X is selected from the group consisting of asingle bond, CH₂, O, S, N(H), and C(O); and n is 0, 1, 2 or 3. In oneembodiment of Formula I, R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each,independently, selected from the group consisting of H, lower alkoxy,hydroxyl, phosphate and phosphoramidate. In another embodiment ofFormula I, X is a single bond.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula II:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindicate a single or double bond; R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, halogen,lower alkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate,and amino acid acyl group; X is selected from the group consisting of asingle bond, CH₂, O, S, N(H), and C(O); and n is 0, 1, 2 or 3. Inanother embodiment of Formula II, R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, loweralkoxy, hydroxyl, phosphate and phosphoramidate. In yet anotherembodiment of Formula I, X is a single bond. In another embodiment ofFormula II, R₁, R₂ and R₃, are each, independently, selected from thegroup consisting of H, OCH₃, phosphate and OH. In still anotherembodiment of Formula II, R_(4,) R₅ and R₆, are each, independently,selected from the group consisting of H, OCH₃, phosphate and OH. Instill another embodiment of Formula II, R₇ is H.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IIa:

or a pharmaceutically acceptable salt thereof,

wherein the phenyl ring “Z” is bonded to either carbon “a” or “b”;

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, phosphate, and hydroxyl;

X is selected from the group consisting of a single bond and C(O); and

n is 1, 2, 3 or 4.

In one embodiment of formula IIa, R₄ and R₅ are OCH₃. In anotherembodiment of formula IIa, n is 1. In another embodiment of formula IIa,n is 3. In another embodiment of formula IIa, n is 4.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IIb:

or a pharmaceutically acceptable salt thereof,

wherein the phenyl ring “Z” is bonded to either carbon “a” or “b”;

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, phosphate, and hydroxyl;

X is selected from the group consisting of a single bond and C(O); and

n is 1, 2, 3 or 4.

In one embodiment of formula IIb, R₄ is H or OH, and R₅ is OH. Inanother embodiment of formula IIb, R₄ is H or phosphate, and R₅ isphosphate. In another embodiment of formula IIb, n is 1. In anotherembodiment of formula IIb, n is 3. In another embodiment of formula IIb,n is 4.

In particular embodiments of formula IIa or IIb, R₁ and R₃ are H. Inother embodiments of formula Ia or IIb, R₁ is OH and R₃ is H. In otherembodiments of formula IIa or IIb, R₁ is phosphate and R₃ is H. In yetother embodiments of formula IIa or IIb, R₁ and R₃ are OCH₃.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula III:

or a pharmaceutically acceptable salt thereof, wherein the dashed lineindicates a single or double bond; X is selected from the groupconsisting of a single bond, CH₂, O, S, N(H), and C(O); and R¹, R₂, R₃,R⁴, R₅, R₆ and R₇ are each, independently, selected from the groupconsisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl, amine,phosphate, phosphoramidate, and amino acid acyl group. In one embodimentof Formula III, X is a single bond, and R₁, R₂ and R₃, are each,independently, selected from the group consisting of H, OCH₃, phosphateand OH. In another embodiment of Formula III, X is a single bond, andR₄, R₅ and R₆, are each, independently, selected from the groupconsisting of H, OCH₃, phosphate and OH.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula IV:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindependently indicate a single or double bond; X is selected from thegroup consisting of a single bond, CH₂, O, S, N(H), and C(O); R₁, R₂,R₃, R₄, R₅, R₆ and R₇ are each, independently, selected from the groupconsisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl, amine,phosphate, phosphoramidate, and amino acid acyl group; and phenyl ring“Z” is bonded to either carbon “a” or “b.” In another embodiment ofFormula IV, the dashed lines are single bonds, X is a single bond, andR₁, R₂ and R₃, are each, independently, selected from the groupconsisting of H, OCH₃, phosphate and OH. In still another embodiment ofFormula IV, the dashed lines are single bonds, X is a single bond, andR₄, R₅ and R₆, are each, independently, selected from the groupconsisting of H, OCH₃, phosphate and OH. In still another embodiment ofFormula IV, the dashed lines are single bonds, X is a single bond, andthe phenyl ring “Z” is bonded to carbon “a.” In yet another embodimentof Formula IV, the dashed lines are single bonds, X is a single bond,and phenyl ring “Z” is bonded to carbon “b.”

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IVa:

or a pharmaceutically acceptable salt thereof,wherein the phenyl ring “Z” is bonded to either carbon “a” or “b”; and

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, phosphate, and hydroxyl.

In one embodiment of formula IVa, R₁ and R₃ are OCH₃. In anotherembodiment of formula IVa, R₄ and R₅ are OH. In another embodiment offormula IVa, R₄ and R₅ are phosphate. In yet another embodiment offormula IVa, phenyl ring “Z” is bonded to carbon “a.”

In another general aspect, the present invention provides aCombretastatin analog of the following general formula IVb:

or a pharmaceutically acceptable salt thereof, wherein the phenyl ring“Z” is bonded to either carbon “a” or “b”; and

R₁, R₃, R₄ and R₅ are each, independently, selected from the groupconsisting of H, lower alkoxy, phosphate, and hydroxyl.

In one embodiment of formula IVb, R₁ and R₃ are OCH₃. In anotherembodiment of formula IVb, R₄ and R₅ are OH. In another embodiment offormula IVb, R₄ and R₅ are phosphate. In yet another embodiment offormula IVb, phenyl ring “Z” is bonded to carbon “a.”

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula V:

or a pharmaceutically acceptable salt thereof, wherein R₆ is selectedfrom the group consisting of H, halogen, lower alkyl, lower alkoxy,hydroxyl, amine, phosphate, phosphoramidate, and amino acid acyl group.In another embodiment of Formula V, R₆ is selected from the groupconsisting of H, OCH₃, phosphate and OH. In yet another embodiment ofFormula V, R₆ is OH.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula VI:

or a pharmaceutically acceptable salt thereof, wherein R₁, R₃, R₅ and R₆are each, independently, selected from the group consisting of H,halogen, lower alkyl, lower alkoxy, hydroxyl, amine, phosphate,phosphoramidate, and amino acid acyl group; and phenyl ring “Z” isbonded to either carbon “a” or “b.” In one aspect of Formula VI, R₁ andR₃ are selected from the group consisting of H, OCH₃, phosphate and OH.In another aspect of Formula VI, R₅ and R₆, are each, independently,selected from the group consisting of H, OCH₃, phosphate and OH. Instill another aspect of Formula VI, the phenyl ring “Z” is bonded tocarbon “a.” In another aspect of Formula VI, the phenyl ring “Z” isbonded to carbon “b.”

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula VII:

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R₄,R₅, R₆, and R₇ are each, independently, selected from the groupconsisting of OH, phosphate and OCH₃.

In another general aspect, the present invention provides aCombretastatin analog of the following general formula VIII:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindicate a single or double bond; R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, halogen,lower alkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate,and amino acid acyl group; X is selected from the group consisting of asingle bond, CH₂, O, S, N(H), and C(O); X₁, X₂, X₃, X₄ and X₅ are each,independently, selected from the group consisting of C, C(H), N, N(H), Oand S, provided that at least one of X₁, X₂, X₃, X₄ and X₅ is not C orC(H); and n is 0, 1, 2 or 3. In one embodiment of Formula I, R₁, R₂, R₃,R₄, R₅, R₆ and R₇ are each, independently, selected from the groupconsisting of H, lower alkoxy, hydroxyl, phosphate and phosphoramidate.In another embodiment of Formula I, X is a single bond.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula IX:

or a pharmaceutically acceptable salt thereof, wherein the dashed lineindicates a single or double bond; X is selected from the groupconsisting of a single bond, CH₂, O, S, N(H), and C(O); X₁, X₂, X₃, X₄and X₅ are each, independently, selected from the group consisting of C,C(H), N, N(H), O and S, provided that at least one of X₁, X₂, X₃, X₄ andX₅ is not C or C(H); R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are each,independently, selected from the group consisting of H, halogen, loweralkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate, andamino acid acyl group, and ring “Z” is bonded to either carbon “a” or“b.” In one embodiment of Formula III, X is a single bond, and R₁, R₂and R₃, are each, independently, selected from the group consisting ofH, OCH₃, phosphate and OH. In another embodiment of Formula III, X is asingle bond, and R₄, R₅ and R₆, are each, independently, selected fromthe group consisting of H, OCH₃, phosphate and OH.

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula X:

or a pharmaceutically acceptable salt thereof, wherein R₁, R₃, R₅ and R₆are each, independently, selected from the group consisting of H,halogen, lower alkyl, lower alkoxy, hydroxyl, amine, phosphate,phosphoramidate, and amino acid acyl group; X₁, X₂, X₃, X₄ and X₅ areeach, independently, selected from the group consisting of C, C(H) andN, provided that at least one of X₁, X₂, X₃, X₄ and X₅ is not C or C(H);and phenyl ring “Z” is bonded to either carbon “a” or “b.” In one aspectof Formula VI, R₁ and R₃ are selected from the group consisting of H,OCH₃, phosphate and OH. In another aspect of Formula VI, R₅ and R₆, areeach, independently, selected from the group consisting of H, OCH₃,phosphate and OH. In still another aspect of Formula VI, the phenyl ring“Z” is bonded to carbon “a.” In another aspect of Formula VI, the phenylring “Z” is bonded to carbon “b.”

In another general aspect, the present invention provides aCombretastatin analog of the following general Formula XI:

or a pharmaceutically acceptable salt thereof, wherein R₁, R₄, R₅ and R₆are each, independently, selected from the group consisting of OH,phosphate, and OCH₃ and X₁, X₂, X₃, X₄ and X₅ are each, independently,selected from the group consisting of C, C(H) and N, provided that atleast one of X₁, X₂, X₃, X₄ and X₅ is not C or C(H).

In another embodiment, the invention includes any novel compound orpharmaceutical compositions containing compounds of the inventiondescribed herein. For example, compounds and pharmaceutical compositionscontaining compounds set forth herein (e.g., Table I and Table II) arepart of this invention, including prodrugs thereof and salts thereof,e.g., pharmaceutically acceptable salts. Like compounds of Formulas I,II, IIa, IIb, III, IV, IVa, IVb, V, VI, VII, VIII, IX, X and XII, thecompounds of Table I and Table II are also considered to be “compoundsof the invention.”

Particular compounds of the invention also include the followingcompounds of Table I and Table II, each of which is considered aseparate embodiment of the invention. The centroid to centroid distanceis included in brackets after the name of the compound.

TABLE I

TABLE II

(15) (1-Hydroxy-2-methoxy-8,9-dihydro- (16)(1-Hydroxy-2-methoxy-8,9-dihydro- 7H-benzocyclohepten-6-yl)-(3,4,5-7H-benzocyclohepten-5-yl)-(3,4,5- trimethoxy-phenyl)-methanone [6.71 Å]trimethoxy-phenyl)-methanone [6.64 Å]

(17) (1-Hydroxy-2-methoxy-7,8,9,10- (18) (1-Hydroxy-2-methoxy-7,8,9,10-tetrahydro-benzocycloocten-6-yl)-(3,4,5-tetrahydro-benzocycloocten-5-yl)-(3,4,5- trimethoxy-phenyl)-methanone[6.76 Å] trimethoxy-phenyl)-methanone [10.01 Å]

(19) (2,3-Dihydroxy-4-methoxy-phenyl)- (20)(3-Hydroxy-4-methoxy-phenyl)- (4,5,6-trimethoxy-3H-inden-1-yl)-(4,5,6-trimethoxy-3H-inden-1-yl)- methanone [5.28 Å] methanone [5.15 Å]

(21) (2,3-Dihydroxy-4-methoxy-phenyl)- (22)(3-Hydroxy-4-methoxy-phenyl)- (5,6,7-trimethoxy-3,4-dihydro-naphthalen-(5,6,7-trimethoxy-3,4-dihydro-naphthalen- 1-yl)-methanone [5.18 Å]1-yl)-methanone [5.03 Å]

(23) (2,3-Dihydroxy-4-methoxy-phenyl)- (24)(3-Hydroxy-4-methoxy-phenyl)- (1,2,3-trimethoxy-8,9-dihydro-7H-(1,2,3-trimethoxy-8,9-dihydro-7H- benzocyclohepten-5-yl)-methanone [6.67Å] benzocyclohepten-5-yl)-methanone [6.67 Å]

(25) (2,3-Dihydroxy-4-methoxy-phenyl)- (26)(3-Hydroxy-4-methoxy-phenyl)- (1,2,3-trimethoxy-7,8,9,10-tetrahydro-(1,2,3-trimethoxy-7,8,9,10-tetrahydro- benzocycloocten-5-yl)-methanone[6.69 Å] benzocycloocten-5-yl)-methanone [6.68 Å]

Compounds of the inventions can be synthesized according to standardorganic synthesis procedures that are known in the art. Synthesisprocedures for the compounds of the invention are also described in theExperimental section and Drawings included herewith.

Acid addition salts of the compounds of the invention are most suitablyformed from pharmaceutically acceptable acids, and include for examplethose formed with inorganic acids e.g. hydrochloric, sulphuric orphosphoric acids and organic acids e.g. succinic, maleic, acetic orfumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalatesmay be used for example in the isolation of the compounds of Formulas I,II, IIa, IIb, III, IV, IVa, IVb, V, VI, VII, VIII, IX, X and XII, andthe compounds of the invention for laboratory use, or for subsequentconversion to a pharmaceutically acceptable acid addition salt. Alsoincluded within the scope of the invention are solvates and hydrates ofthe invention.

The conversion of a given compound salt to a desired compound salt isachieved by applying standard techniques, in which an aqueous solutionof the given salt is treated with a solution of base e.g. sodiumcarbonate or potassium hydroxide, to liberate the free base which isthen extracted into an appropriate solvent, such as ether. The free baseis then separated from the aqueous portion, dried, and treated with therequisite acid to give the desired salt.

In vivo hydrolyzable esters or amides of certain compounds of theinvention can be formed by treating those compounds having a freehydroxy or amino functionality with the acid chloride of the desiredester in the presence of a base in an inert solvent such as methylenechloride or chloroform. Suitable bases include triethylamine orpyridine. Conversely, compounds of the invention having a free carboxygroup may be esterified using standard conditions which may includeactivation followed by treatment with the desired alcohol in thepresence of a suitable base.

III) Treatment of Cancer and Other Malignant Proliferative Disorders

The Combrestatin analogs of the present invention demonstrate remarkablecytotoxicity against a variety of human cancer cell lines. The abilityof an agent to inhibit tubulin assembly and microtubule formation is animportant property of many anticancer agents. Disruption of microtubulesthat comprise the cytoskeleton and mitotic spindle apparatus caninterfere dramatically with the ability of a cell to successfullycomplete cell division.

The compounds of the present invention are highly cytotoxic to activelyproliferating cells, inhibiting their mitotic division and often causingtheir selective apoptosis while leaving normal quiescent cellsrelatively unaffected. Accordingly, the antiproliferative oranti-mitotic properties of the compounds of the present invention can beused to directly inhibit the proliferation of, or impart directcytotoxicity towards, the cells of malignant or neoplastic tumors orcancers including:

-   -   1) carcinomas, such as those of the bladder, breast, colon,        rectum, kidney, liver, lung (including small cell lung cancer),        pharynx, esophagus, gall bladder, urinary tract, ovaries,        cervix, uterus, pancreas, stomach, endocrine glands (including        thyroid, adrenal, and pituitary), prostate, testicles and skin,        including squamous cell carcinoma;    -   2) hematopoietic tumors of lymphoid lineage, including leukemia,        acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell        lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins        lymphoma, hairy cell lymphoma and Burkett's lymphoma;    -   3) hematopoietic tumors of myeloid lineage, including acute and        chronic myelogenous leukemias, myelodysplastic syndrome and        promyelocytic leukemia;    -   4) tumors of mesenchymal origin, including fibrosarcoma and        rhabdomyosarcoma;    -   5) tumors of the central and peripheral nervous system and        meninges, including astrocytoma, neuroblastoma, glioma,        schwannomas, retinoblastomas, neuroma, glioma, glioblastoma; and    -   6) other tumors, including melanoma, seminoma, teratocarcinoma,        osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid        follicular cancer, anaplastic thyroid cancer and Kaposi's        sarcoma.

Alternatively, the compounds of the present invention can impartindirect control of the growth and proliferation of the above tumors andcancers due to their effects on malignant proliferating vasculature,such as the endothelium, arteries, blood vessels, or neovasculatureformed by a tumor. These antivascular properties include, but are notlimited to, the selective destruction, damage, or occlusion, whetherreversible or irreversible, partial or complete, of proliferating tumorvasculature.

The compounds of the present invention may also be useful for thetreatment of the tumors and cancer described above when used eitheralone or in combination with radiotherapy and/or other chemotherapeutictreatments conventionally administered to patients for treating solidtumor cancers. For example, compounds of the present invention may beadministered with chemotherapeutic agents selected from one of thefollowing mechanistic classes:

-   -   1. Alkylating agents: compounds that donate an alkyl group to        nucleotides. Alkylated DNA is unable to replicate itself and        cell proliferation is stopped. Exemplary alkylating agents        include Melphalan, Chlorambucil, cyclophosphamide, ifosfamide,        busulfan, dacarbaine, methotrexate, 5-FU, cytosine arabinsoide,        or 6-thioguanine.    -   2. Antianziozenic agents: compounds that inhibit the formation        of tumor vasculature. Exemplary anti-angiogenic agents include        TNP-470 or Avastin™.    -   3. Antitumor Antibiotics: compounds having antimicrobial and        cytotoxic activity.        -   Such compounds also may interfere with DNA by chemically            inhibiting enzymes and mitosis or altering cellular            membranes. Exemplary anti-tumor antibiotics include            Actinomycin-D, bleomycin, mitomycin-C, Dactinomycin,            Daunorubicin, and Doxorubicin.    -   4. Topoisomerase Inhibitors: agents which interfere with        topoisomerase activity thereby inhibiting DNA replication. Such        agents include CPT-11 and Topotecan.    -   5. Hormonal Therapy: includes, but is not limited to        anti-estrogens. An exemplary antiestrogen is Tamoxifen.    -   6. Antimicrotubule compounds. The compounds of the invention may        be used in combination with other antimicrotubule compounds,        including for example, Combretastatin A-1 Diphosphate,        Combretastatin A-4 Phosphate, Vincristine, paclitaxel, taxotere,        etoposide, and vinblastine.

In yet other embodiments, the compounds of the present invention may beadministered with a platinum coordination compounds (e.g carboplatin,cisplatin, or oxaliplatin).

IV) Treatment of Nonmalignant Vascular Proliferative Disorders

In other embodiments, the compounds of the invention as well as analogsthereof, may be employed as vascular targeting agents (VTAs), and thusare also useful for the treatment of non-malignant vascularproliferative disorders, where the endothelium, artery, blood vessel, orneovasculature is not associated with a tumor but is nonetheless formedby undesirable or pathological angiogenesis. Such disease statesinclude, without limitation:

-   -   1) ocular diseases such as wet or age-related macular        degeneration, myopic macular degeneration, diabetic retinopathy,        retinopathy of prematurity, diabetic macular edema, uveitis,        neovascular glaucoma, rubeosis, retrolental fibroplasias,        angioid streaks, ocular histoplasmosis, and corneal        neovascularization;    -   2) inflammatory disorders such as endometriosis, psoriasis,        rheumatoid arthritis, Osler-Webber Syndrome, wound granulation,        and    -   3) cardiovascular diseases such as atherosclerosis, atheroma,        restenosis, haemangioma, restenosis.

In one preferred embodiment, the present invention is directed to theadministration of compound of the invention for the treatment ofnon-malignant vascular proliferative disorders in the retinal tissue ofthe eye. Neovascularization of retinal tissue or “retinopathy” is apathogenic condition characterized by vascular proliferation and occursin a variety of ocular diseases with varying degrees of vision failure.The blood-retinal barrier (BRB) is composed of specializednonfenestrated tightly-joined endothelial cells that form a transportbarrier for certain substances between the retinal capillaries and theretinal tissue. The nascent vessels of the retina associated with theretinopathies are aberrant, much like the vessels associated with solidtumors. Tubulin binding agents, inhibitors of tubulin assembly, andvascular targeting agents may be able to attack the aberrant vesselsbecause these vessels do not share architectural similarities with theBRB. Tubulin binding agents may halt the progression of the disease muchlike they do with a tumor-vasculature. The administration of a VTA forthe pharmacological control of the retinal neovascularization associatedwith retinopathies as wet macular degeneration, proliferative diabeticretinopathy or retinopathy of prematurity, would potentially benefitpatients for which few therapeutic options are available.

The compounds of the present invention are also contemplated for use inthe treatment of vascular disease, particularly atherosclerosis andrestenosis. Atherosclerosis is the most common form of vascular diseaseand leads to insufficient blood supply to critical body organs,resulting in heart attack, stroke, and kidney failure. Additionally,atherosclerosis causes major complications in those suffering fromhypertension and diabetes, as well as tobacco smokers. Atherosclerosisis a form of chronic vascular injury in which some of the normalvascular smooth muscle cells (VSMC) in the artery wall, which ordinarilycontrol vascular tone, regulate blood flow, change their nature anddevelop “cancer-like” behavior. These VSMC become abnormallyproliferative, secreting substances (growth factors, tissue-degradationenzymes and other proteins) which enable them to invade and spread intothe inner vessel lining, blocking blood flow and making that vesselabnormally susceptible to being completely blocked by local bloodclotting, resulting in the death of the tissue served by that artery.

Restenosis, the recurrence of stenosis or artery stricture aftercorrective surgery, is an accelerated form of atherosclerosis. Recentevidence has supported a unifying hypothesis of vascular injury in whichcoronary artery restenosis along with coronary vein graft and cardiacallograft atherosclerosis can be considered to represent amuch-accelerated form of the same pathogenic process that results inspontaneous atherosclerosis. Restenosis is due to a complex series offibroproliferative responses to vascular injury involving potentgrowth-regulatory molecules, including platelet-derived growth factor(PDGF) and basic fibroblast growth factor (bFGF), also common to thelater stages in atherosclerotic lesions, resulting in vascular smoothmuscle cell proliferation, migration and neointimal accumulation.

Restenosis occurs after coronary artery bypass surgery (CAB),endarterectomy, and heart transplantation, and particularly after heartballoon angioplasty, atherectomy, laser ablation or endovascularstenting (in each of which one-third of patients redevelop restenosiswithin 6 months), and is responsible for recurrence of symptoms (ordeath), often requiring repeat revascularization surgery. Despite over adecade of research and significant improvements in the primary successrate of the various medical and surgical treatments of atheroscleroticdisease, including angioplasty, bypass grafting and endarterectomy,secondary failure due to late restenosis continues to occur in 30-50% ofpatients. Repeated revascularization surgery consumes time and money, isinconvenient to the patient, and can carry a significant risk ofcomplications or death. The most effective way to prevent restenosis isat the cellular level.

V) Dosage and Administration of Compounds

A typical daily dose will contain from about 0.1 mg/kg to about 1000mg/kg of the active compound of this invention. Preferably, daily doseswill be about 10 mg/kg to about 100 mg/kg, and most preferably about 10mg.

In effecting treatment of a patient afflicted with a condition, diseaseor disorder described herein, a compound of the present invention can beadministered systemically in any form or mode which makes the compoundbioavailable in effective amounts. Systemic administration may beaccomplished by administration of a compound of the present inventioninto the bloodstream at a site which is separated by a measurabledistance from the diseased or affected organ or tissue. For example,compounds of the present invention can be administered orally,parenterally, subcutaneously, intramuscularly, intravenously,transdermally, intranasally, rectally, buccally, and the like. Oral orintravenous administration is generally preferred for treatingneoplastic disease or cancer. Alternatively, the compound may beadministered non-systemically by local administration of the compound ofthe present invention directly at the diseased or affected organ ortissue. Treatment of ocular diseases characterized by the presence ofnon-malignant proliferative vasculature or neovascularization, can beachieved using non-systemic administration methods such as intravitrealinjection, sub-Tenon's injection, ophthalmic drops, iontophoresis,topical formulation and implants and/or inserts. One skilled in the artof preparing formulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of thecompound selected, the disease state to be treated, the stage of thedisease, and other relevant circumstances.

It will be understood by the skilled reader that all of the compoundsused in the present invention are capable of forming salts, and that thesalt forms of pharmaceuticals are commonly used, often because they aremore readily crystallized and purified than are the free bases. In allcases, the use of the pharmaceuticals described above as salts iscontemplated in the description herein, and often is preferred, and thepharmaceutically acceptable salts of all of the compounds are includedin the names of them.

According to another aspect, the present invention provides apharmaceutical composition, which comprises a compound of the presentinvention or a pharmaceutically acceptable salt thereof as definedhereinabove and a pharmaceutically acceptable diluent or carrier.

The pharmaceutical compositions are prepared by known procedures usingwell-known and readily available ingredients. In making the compositionsof the present invention, the active ingredient will usually be mixedwith a carrier, or diluted by a carrier, or enclosed within a carrier,and may be in the form of a capsule, sachet, paper, or other container.When the carrier serves as a diluent, it may be a solid, semi-solid, orliquid material which acts as a vehicle, excipient, or medium for theactive ingredient. The compositions can be in the form of tablets,pills, powders, lozenges, sachets, cachets, elixirs, suspensions,emulsions, solutions, syrups, aerosols, ointments containing, forexample, up to 10% by weight of active compound, soft and hard gelatincapsules, suppositories, sterile injectable solutions, and sterilepackaged powders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum, acacia,calcium phosphate, alginates, tragcanth, gelatin, calcium silicate,micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propyl hydroxybenzoates, talc,magnesium stearate, and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents, or flavoring agents.Compositions of the invention may be formulated so as to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well know in theart.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 1 mg to about 500 mg, more preferably about5 mg to about 300 mg (for example 25 mg) of the active ingredient. Theterm “unit dosage form” refers to a physically discrete unit suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical carrier, diluent, or excipient.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way.

The compositions of the present invention may be formulated in aconventional manner using one or more pharmaceutically acceptablecarriers. Thus, the active compounds of the invention may be formulatedfor oral, buccal, transdermal (e.g., patch), intranasal, parenteral(e.g., intravenous, intramuscular or subcutaneous) or rectaladministration or in a form suitable for administration by inhalation orinsufflation.

Alternatively, compounds of the present invention can be administered inthe form of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The compositions in liposome form can contain, in addition to acompound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidylcholines (lecithins), both natural and synthetic.Methods to form liposomes are well known in the art (see for example,Prescott Ed., Methods in Cell Biology, Volume XTV, Academic Press, NewYork, N.Y., 1976, p 33).

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose of calcium phosphate); lubricants (e.g., magnesium stearate,talc or silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically A acceptable additives such as suspending agents (e.g.,sorbitol syrup, methyl cellulose or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters or ethyl alcohol); and preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

For buccal administration the composition may take the form of tabletsor lozenges formulated in conventional manner.

The active compounds of the invention may be formulated for parenteraladministration by injection, including using conventionalcatheterization techniques or infusion. Formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulating agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in a powder form for reconstitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The active compounds of the invention may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

For intranasal administration or administration by inhalation, theactive compounds of the invention are conveniently delivered in the formof a solution or suspension from a pump spray container that is squeezedor pumped by the patient or as an aerosol spray presentation from apressurized container or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. The pressurized containeror nebulizer may contain a solution or suspension of the activecompound. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated containing a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

Tablets or capsules of the compounds may be administered singly or twoor more at a time as appropriate. It is also possible to administer thecompounds in sustained release formulations.

The physician will determine the actual dosage which will be mostsuitable for an individual patient and it will vary with the age, weightand response of the particular patient. The above dosages are exemplaryof the average case. There can of course, be individual instances wherehigher or lower dosage ranges are merited, and such are within the scopeof this invention.

The compounds of the present invention can be administered by inhalationor in the form of a suppository or pessary, or they may be appliedtopically in the form of a lotion, solution, cream, ointment or dustingpowder. An alternative means of transdermal administration is by use ofa skin patch. For example, they can be incorporated into a creamconsisting of an aqueous emulsion of polyethylene glycols or liquidparaffin. They can also be incorporated, at a concentration of between 1and 10% by weight, into an ointment consisting of a white wax or whitesoft paraffin base together with such stabilizers and preservatives asmay be required.

“Administering” means any of the standard methods of administering acompound to a subject, known to those skilled in the art. Examplesinclude, but are not limited to intravenous, intramuscular orintraperitoneal administration.

For the purposes of this invention “pharmaceutically acceptablecarriers” means any of the standard pharmaceutical carriers. Examples ofsuitable carriers are well known in the art and may include, but are notlimited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solutions, phosphate buffered salinecontaining POLYSORB 80, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Other carriers may also include sterilesolutions, tablets, coated tablets, and capsules.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gums glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known convention methods.

Methods of determining an “effective amount” are well known to thoseskilled in the art and depend upon factors including, but not limitedto, the size of the patient and the carrier used.

The invention is further defined by reference to the following examplesand preparations which describe the manner and process of making andusing the invention and are illustrative rather than limiting. It willbe apparent to those skilled in the art that many modifications, both tothe materials and methods, may be practiced without departing from thepurpose and interest of the invention.

EXAMPLES

Materials and Methods

Chemicals were commercially obtained from the Aldrich Chemical Company,Fisher Scientific and ACROS Chemicals and used directly as purchased.Solvents such as acetone, diethylether and ethylacetate were used aspurchased, and other solvents were purified by standard procedures.Tetrahydrofuran (THF) was dried over potassium metal and benzophenoneand distilled freshly prior to use; methylene chloride (CH₂Cl₂) wasdried using calcium hydride and distilled prior to use. Triethyl aminewas distilled over calcium hydride and stored in a sealed bottle.

Reactions were followed by thin layer chromatography (TLC) and/or gaschromatography. Purification of products was carried out using flashcolumn chromatography with silica gel. Silica gel plates for thin layerchromatography and silica gel (260-400 mesh) for column chromatographywere obtained from Merck EM Science.

¹H and ¹³C NMR spectra were recorded in deuterated chloroform ordeuterated methyl sulfoxide or deuterium oxide using an AMX 360 MHz (90MHz for ¹³C, and 145 MHz for ^(3I)P) or a DPX Avance 300 MHz (75 MHz for¹³C, and 120 MHz for ³¹P) Briiker NMR spectrometer. Peaks are listed assinglet (s), doublet (d), doublet of doublet (dd), triplet (t), ormultiplet (m) with the coupling constant (J) expressed in Hz.High-resolution mass spectra were obtained using a VG/Fisons GC/NASSHigh Resolution Mass Spectrometer. Elemental analyses were obtained fromAtlantic Microlab Inc., Norcross, Ga. Melting points were determinedusing a Thomas-Hoover melting point apparatus and are uncorrected.

In many of the reactions, particularly those using Pd, methanol may besubstituted for ethanol, and vice versa. Additionally, AlCl₃ may besubstituted for TiCl₄, and vice versa.

Example 1 Synthesis of Representative Combretastatin Analogs Synthesisof3-Methoxy-9-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene(2; see FIG. 1) 6-Methoxy-1-methylene-1,2,3,4-tetrahydro-naphthalene(27)

To a dry 500 mL 3-necked round bottom flask equipped with a refluxcondenser and magnetic stir bar, was charged sodium hydride (3.0 g,172.89 mmol). The reaction flask was put under nitrogen, and 35 mL ofanhydrous DMSO was added. The reaction mixture was heated to 70-75° C.,and stirred at that temperature until the evolution of hydrogen ceased.The reaction mixture was cooled to room temperature and additional 25 mLof DMSO was added. Methyltriphenylphosphonium iodide (46.57 g, 115.26mmol) was added in portions over a period of 1 h. 25 mL of additionalDMSO was added to facilitate easy stirring. After the completion ofaddition, the reaction mixture was stirred for 20 minutes.6-methoxytetralone (10.156 g, 57.63 mmol) dissolved in 10 mL ofanhydrous DMSO was added to reaction mixture. Then, the reaction mixturewas heated to 60-65° C., and stirred at that temperature for 8 h. Thereaction mixture was poured into a 500 mL Erlenmeyer flask containing150 mL of crushed ice and 150 mL of hexanes. The resulting mixture wasstirred vigorously for 15 min, and then extracted with hexanes. Thecombined organic layers were washed DMSO:Water (1:1) and then dried overanhydrous sodium sulfate, filtered and evaporated under reducedpressure. Purification by flash column chromatography (silica gel 3:97EtOAc:Hexanes) yielded 9.42 g of 27 as white solid (94%). Rf: 0.71,(30:70, EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 7.8 (d, J=8.73 Hz, 1H), δ 6.74 (dd, J=8.72Hz, 2.74 Hz, 1 H), δ 6.62 (d, J=2.71 Hz, 1 H), δ 5.34 (s, 1H), δ 4.84(s, 1 H), δ3.76 (s, 3H), δ 2.81 (t, J=6.27 Hz, 2 H), δ 2.53 (t, J=6.34,2H), δ 1.87 (p, J=6.16, 2 H).

2-Methoxy-5,7,8,9-tetrahydro-benzocyclohepten-6-one (28)

2-Methoxy-5,7,8,9-tetrahydro-benzocyclohepten-6-one (28) was preparedaccording to the methods of Miller et al. (Miller et al. J. Org. Chem.1978, 43 (8), 1569) and as outlined below.

Preparation of cyanogen azide: Finely powdered cyanogen azide (8.58 g,132 mmol) was added rapidly to a 0° C. solution of cyanogen bromide(13.98 g, 132 mmol) in 40 mL of anhydrous acetonitrile. The reactionmixture was stirred for 4 h at 0° C. and clear supernatant solutioncontaining cyanogen azide (7.18 g, 105.61 mmol) was filtered and usedfor the ring expansion reaction.

Ring expansion reaction: The exocyclic olefin 27 (4.6 g, 26.40 mmol) wascharged in a 250 mL round bottom flask under nitrogen. 40 mL ofanhydrous methanol:acetonitrile (1:1) was added to the reaction flask,and the reaction mixture was stirred at room temperature. To this wasadded freshly prepared cyanogen azide (7.18 g, 105.61 mmol) and thereaction mixture was stirred at room temperature for 48 h. 25 mL of 6 Mof HCl was added and the reaction was stirred at 50° C. for 4 h. Thereaction mixture was then cooled to room temperature and extracted withether (2×200 mL). The ethereal extracts were washed with water untilneutral and then dried over anhydrous sodium sulfate. The organic phasewas then percolated through a column of basic alumina capped with alayer of celite to remove the explosive azides. Evaporation of thesolvent followed by purification by flash column chromatography (8:92EtOAc:Hexanes) yielded 3.33 g of 28 (off-white solid on refrigeration).Rf. 0.38 (30:70, EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 7.06 (d, J=7.72 Hz, 1H), δ 6.70 (m, 2 H), δ3.79 (s, 3 H), δ 3.65 (s, 2 H), δ 2.90 (m, 2H), δ 2.55 (t, J=6.8, 2 H),δ1.98 (m, 2H).

2-Methoxy-6,7,8,9-tetrahydro-5H-benzocycloheptane(29)

Amalgamated zinc was prepared by shaking 4 g of Zn powder and 400 mg ofmercuric chloride, 4 mL of water and 0.25 mL of con. HCl in a 100 mLround bottom flask for 10 min. The supernatant liquid was decanted and28 (215 mg, 1.13 mmol) was added, followed by 15 mL of con. HCl. Thereaction mixture was then refluxed for 3 hours. The reaction mixture wascooled and extracted with ether (20 mL×3) and the combined etherealextracts were washed with water until neutral, then dried over anhydrousmagnesium sulfate, filtered and evaporated under reduced pressure. Thecompound was purified by flash column chromatography on neutral alumina.Product, 29 was obtained in just hexanes (140 mg, 74%). Rf value: 0.535(10:90, EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 6.99 (d, J=8.12 Hz, 1H), δ 6.67 (d, J=2.68Hz, 1 H), δ 6.62 (dd, J=8.12 Hz, 2.74 Hz, 1 H), δ 3.76 (s, 3 H), δ 2.73(m, 2 H), δ 1.79 (m, 2H), δ 1.62 (m, 4 H).

2-Methoxy-6,7,8,9-tetrahydro-benzocyclohepten-5-one (30)

29 (3.42 g, 19.4 mmol) was taken in a 500 mL round bottom flask, and tothis was added 40 mL of glacial acetic acid followed by a CrO₃ (5.82 g,58.21 mmol) dissolved in 5 mL of water and 20 mL of acetic aciddropwise. After the addition was completed, the reaction mixture wasstirred for 24 h at room temperature. 100 mL of water was added to thereaction mixture and then extracted with ether (100×3). The combinedethereal extracts were then washed with 5% NaOH solution, until theaqueous phases were alkaline. The organic phase was washed with wateruntil neutral and then dried over anhydrous magnesium sulfate, filteredand evaporated under reduced pressure. Purification by flash columnchromatography (neutral alumina) afforded 910 mg (25%) of product ascolorless oil. Product obtained in 10:90 (EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 7.79 (d, J=8.58 Hz, 1H), 6.81 (dd, J=8.70 Hz,2.66 Hz, 1 H), δ 6.70 (d, J=2.68 Hz, 1 H), δ 3.84 (s, 3 H), δ 2.91(m, 2H), δ 2.72 (m, 2H), δ 1.84 (m, 4 H).

2-Methoxy-5-(3,4,5-trimethoxyphenyl)-6,7,8,9-tetrahydro-benzocyclohepten-5-ol(32)

200 mL of anhydrous ether was, added to 3,4,5-trimethoxybromobenzene(1.23 g, 4.97 mmol) in a 500 mL round bottom flask under nitrogen. Thetemperature of the reaction mixture was brought to −78° C.n-Butyllithium (4.5 mL, 11.25 mmol) was added dropwise. The reactionmixture was then stirred until the temperature was raised gradually to−30° C. 30 (0.86 g, 4.5 mmol) dissolved in 25 mL of dry ether was addeddropwise and the reaction mixture was allowed to stir until thetemperature warmed up to room temperature. 25 mL of water was added andthe product was extracted with ether (3×50), combined organic phaseswere dried over anhydrous magnesium sulfate, filtered and evaporatedunder reduced pressure. Purification by column chromatography (silicagel, 20:80 EtOAc:Hexanes) yielded 800 mg (49%) of 32 as pale yellow oilRf: 0.16 (30:70, EtOAc:Hexanes).

3-Methoxy-9-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene(2)

A mixture of the 32 (800 mg, 2.23 mmol) in 20 mL of acetic acid and 100mL of water, taken in a 250 mL round bottom flask were refluxed for 12h. The reaction mixture was cooled and extracted with dichloromethane(3×25 mL) and the combined organic phases were dried over magnesiumsulfate. The solvent evaporated and the crude product was purified byflash column chromatography (10:90 EtOAc:Hexanes), to afford 690 mg(91%) of 2 as white crystals. Rf: 0.43 (30:70, EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 6.97 (d, J=8.46 Hz, 1H), δ 6.83 (d, J=2.61Hz, 1H), δ6.74 (dd, J=8.49 Hz, 2.73 Hz, 1H), δ 6.49 (s, 2H), δ 6.35 (t,J=7.32 Hz, 1H), δ 3.86 (s, 3H), δ 3.84 (s, 3H), δ 3.80 (s, 3H), δ 2.64(t, J=6.99 Hz, 2H), δ 2.18 (p, J=7.05 Hz, 2H), δ 1.97 (m, 2H).

Synthesis of3-Methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene(1; see FIG. 1)2-hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)benzosuberan (31)

3,4,5-trimethoxybromobenzene (0.91 g, 3.68 mmol) was charged in a 500 mLround bottom flask and put under nitrogen. 70 mL of anhydrous ether wasadded and the temperature was brought to −78° C. n-butyllithium (2.94mL, 7.36 mmol) was added dropwise. The reaction mixture was then stirreduntil the temperature was raised gradually to −30° C. 28 (0.7 g, 3.68mmol) dissolved in 20 mL of dry ether was added dropwise and thereaction mixture was allowed to stir until the temperature warmed up toroom temperature. 25 mL of water was added to the reaction mixture andextracted with ether (3×30 mL), combined organic phases were dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Purification by column chromatography (silica gel, 20:80, EtOAc:Hexanes)yielded 500 mg (29%) of 31 as pale yellow oil.

3-Methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene(1)

A mixture of the 31 (500 mg, 1.4 mmol) in 12 mL of acetic acid and 60 mLof water, taken in a 250 mL round bottom flask were refluxed for 12 h.The reaction mixture was cooled and extracted with dichloromethane (3×20mL) and the combined organic phases were dried over magnesium sulfate.The solvent evaporated and the crude product was purified by flashcolumn chromatography (10:90 EtOAc:Hexanes), to afford 60 mg (13%) of 1as white crystals.

NMR data: ¹H (CDCl₃, 300 MHz): δ 7.17 (d, J=7.87 Hz, 1H), δ 6.73 (m,4H), δ 3.90 (s, 6H), δ 3.87 (s, 3H), δ 3.82 (s, 3H), δ 2.81 (t, J=6.21Hz, 2H), δ 2.64 (t, J=6.60 Hz, 2H), δ 2.20 (p, J=6.16 Hz, 2H).

Synthesis of2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol(3; see FIG. 1) 2-Methoxy-5,6,7,8-tetrahydro-naphthalen-1-ol (33)

2-Methoxy-5,6,7,8-tetrahydro-naphthalen-1-ol (33) was prepared accordingto the methods of Ghatak et al., (Ghatak, et al., Tet. Lett. (2003), 44:4145) and as outlined below.

To a well-stirred solution of 6-methoxy-1,2,3,4-tetrahydronapthalene(14.05 g, 86.67 mmol) in sec-butyllithium (100 mL, 110 mmol) at 0° C.,under nitrogen, was added freshly distilledN,N,N′,N′-tetramethylethylenediamiene (13.61 mL) dropwise. After theaddition was complete, the reaction mixture was stirred at roomtemperature for 1 h. Then, the reaction mixture was cooled again to 0°C. and trimethyl borate (12.55 mL, 110 mmol) was added dropwise and thereaction mixture was stirred for 1 h at room temperature. The reactionmixture was cooled back to 0° C. and 7 mL of glacial acetic acid wasadded dropwise, the reaction mixture was let to cool to 0° C. and then35% wt. hydrogen peroxide in water (15 mL) was added dropwise. Thereaction mixture was then allowed to stir at room temperature for 12 h.Saturated ammonium chloride (100 mL) was added. The organic phase wasseparated and collected, and the aqueous phase was extracted with ether.The combined organic phases were washed with brine (if necessary) anddried over anhydrous sodium sulfate, filtered, concentrated in vacuo.Purification by flash column chromatography (silica gel, 2:98EtOAc:Hexanes) yielded 3.5 g (23%) of 33 along with 9.7 g (63.1%) of7-hydroxy-6-methoxy-(1,2,3,4-tetrahydro)naphthalene (obtained in 5:95EtOAc:Hexanes). Rf value for 33 is 0.48 (15:85 EtOAc:Hexanes), for7-hydroxy-6-methoxy-1-tetrahydronapthalene 0.37.

¹H NMR (CDCl₃, 300 MHz): δ 6.67 (d, J=8.28 Hz, 1H), δ 6.58 (d, J=8.79Hz, 1 H), δ 5.64 (s, 1H), δ 3.85 (s, 3H), δ 2.71 (t, J=3.03, 4H), δ 1.76(m, 4H).

5-Isopropoxy-6-methoxy-1,2,3,4-tetrahydro-naphthalene(34)

5-Isopropoxy-6-methoxy-1,2,3,4-tetrahydro-naphthalene (34) was preparedaccording to the methods of Bringmann et al. (Bringmann et al., J. Org.Chem. (2002), 67 (16), 5595) and according to the methods outlinedbelow.

33 (4.55 g, 25.5 mmol) was charged in a 250 mL round bottom flask,equipped with a reflux condenser, under nitrogen gas. 50 mL of anhydrousacetone was added followed by cesium carbonate (66.55 g, 204.3 mmol).2-bromopropane (23.94 mL, 255.0 mmol) was added and the reaction mixturewas refluxed for 12 h. The solvent was filtered and evaporated in vacuoand purified by flash column chromatography (slilica gel, 1:99EtOAc:Hexanes) to afford 5.2 g (93%) of5-isopropoxy-6-methoxy-1,2,3,4-tetrahydronaphtalene (34). Rf value:0.615 (15:85 EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 6.75d, J=8.34 Hz, 1H), δ 6.69 (s, J=8.37 Hz,1H), δ 4.46 (septet, J=6.18 Hz, 1H), δ 3.80 (s, 3H), δ 2.71 (m, 4H), δ1.74 (m, 4H), δ 1.27 J=6.18)

5-Isopropoxy-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (35)

34 (120 mg, 0.55 mmol) was weighed in a 100 mL round bottom flask and asolution of 5 mL of water:dioxane (5:95) was added and the reaction wasset up under nitrogen. A solution of2,4-dichloro-5,6-dicyanobenzoquinone (0.25 g, 1.09 mmol) dissolved in 5mL of dioxane was added dropwise to the reaction mixture. The reactionmixture was stirred for 12 h. The solid separated was filtered andwashed with ethylacetate. The filtrate was concentrated under reducedpressure and 10 mL of saturated sodium bicarbonate solution was addedand the resulting reaction mixture was extracted with ether (3×20 mL)and the combined organic phases dried over anhydrous sodium sulfate,filtered and evaporated in vacuo. Purification by flash columnchromatography (silica gel, 30:70 EtOAc:Hexanes) yielded 90 mg (71%) of35. Rf value: 0.458 (40:60 EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 7.84 (d, J=8.73 Hz, 1H), δ 6.86 (d, J=8.73Hz, 1H) δ 4.45 (septet, J=6.18 Hz, 1H), δ 3.89 (s, 3H), δ 2.95 (t,J=6.00 Hz, 2H), δ 2.59 (t, J=6.18 Hz, 2H), δ 2.06 (p, J=6.57 Hz, 2H) δ1.28 (d, J=6.18 Hz, 6H).

5-Isopropoxy-6-methoxy-1-methylene-1,2,3,4-tetrahydro-naphthalene (36)

To a dry 250 mL 3-necked round bottom flask equipped with a refluxcondenser and magnetic stir bar was added sodium hydride (1.09 g, 47.4mmol) under nitrogen. 20 mL of anhydrous dimethylsulfoxide was added andthe reaction mixture was heated to 70-75° C. for 30 min, until theevolution of hydrogen ceased. The reaction mixture turned green in colorat this point, and then, the reaction mixture was cooled to roomtemperature and additional 15 mL of DMSO was added.Methyltriphenylphosphonium iodide (12.76 g, 31.57 mmol) was added inportions over a period of 30 min. 15 mL of additional DMSO was added andthe reaction mixture was stirred for 20 minutes at room temperature. 35(3.73 g, 15.8 mmol) dissolved in 5 mL of anhydrous DMSO was added toreaction mixture and the temperature was raised to 60-65° C., andstirred at that temperature for 8 h. The reaction mixture was pouredinto a 250 mL Erlenmeyer flask containing 75 mL ice and 75 mL ofhexanes. The resulting mixture was stirred vigorously for 15 min, andthen extracted with hexanes. The combined organic layers were washedDMSO:Water (1:1) and then dried over anhydrous sodium sulfate, filteredand evaporated under reduced pressure. Purification by flash columnchromatography (silica gel 5:95, EtOAc:Hexanes) yielded 320 mg of 36(10%). Rf −0.632, (30% ethylacetate in hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 7.37 (d, J=8.70 Hz, 1H), δ 6.75 (d, J=8.70Hz, 1H), δ 5.35 (s, 1H), δ 4.85 (s, 1H), δ 4.46 (septet, J=6.18 Hz, 1H),δ 3.83 (s, 3H), δ 2.82 (t, J=6.27 Hz, 2H), δ 2.48 (t, J=6.00 Hz, 2H), δ1.83 (p, J=6.33 Hz, 2H), δ 1.27 (d, J=6.18 Hz, 6H).

1-Isopropoxy-2-methoxy-5,7,8,9-tetrahydro-benzocyclohepten-6-one (37)

1-Isopropoxy-2-methoxy-5,7,8,9-tetrahydro-benzocyclohepten-6-one (37)was prepared according to the methods of McMurry et al. (McMurry et al.,J. Org. Chem. (1973), 38 (16), 2821) and as outlined below.

Preparation of cyanogen azide: Finely powedered cyanogen azide (0.51 g,7.8 mmol) was added rapidly to a 0° C. solution of cynaogen bromide(0.83 g, 7.8 mmol) in 5 mL of anhydrous aceotnitrile. The reactionmixture was stirred for 4 h at 0° C. and clear supernatant solutioncontaining cyanogen azide (0.53 g, 7.8 mmol) was drawn into a syringe tobe used for the ring expansion reaction.

Ring expansion reaction: The exocyclic olefin (320 mg, 1.3 mmol) wascharged in a 50 mL round bottom flask, and put under nitrogen. 5 mL ofmethanol:acetonitrile (1:1) was added to the reaction flask, and thereaction mixture was stirred at room temperature. To this was addedfreshly prepared cyanogen azide (0.53 g, 7.8 mmol) and the reactionmixture was stirred at room temperature for 48 h. 5 mL of 6 M of HCl wasadded and the reaction was stirred at 50° C. for 4 h. The reactionmixture was then cooled to room temperature, extracted with ether (2×30mL). The ethereal extracts were washed with water until neutral and thendried over anhydrous sodium sulfate. The organic phase was thenpercolated through a column of basic alumina capped with a layer ofcelite to remove the explosive azides. Evaporation of the solventfollowed by purification by flash column chromatography (8:92EtOAc:Hexanes) yielded 110 mg (33%) of 37 (off-white solid). Rf: 0.476(70:30 hexanes:ethylacetate).

¹H NMR (CDCl₃, 300 MHz): δ 6.83 (d, J=8.28 Hz, 1H), δ 6.71 (d, J=8.25Hz, 1H), δ 4.43 (septet, J=6.14 Hz, 1H), δ 3.82 (s, 3H), δ 3.64 (s, 2H),δ 3.04 (t, J=6.36 Hz, 2H), δ 2.51 (t, J=6.90 Hz), δ 1.94 (p, J=6.90 Hz,2H), δ 1.28 (d, J=6.15 Hz, 6 H). ¹³C NMR (CDCl₃, 75 MHz): δ 209.58,152.24, 144.47, 134.63, 126.91, 124.28, 109.98, 74.80, 55.67, 49.73,43.27, 25.37, 24.46, 22.58.

1-Isopropoxy-2-methoxy-6-(3,4,5-trimethoxy-phenyl)-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-ol(40)

3,4,5-trimethoxybromobenzene (1.49 g, 6.05 mmol) was dissolved in 200 mLof dry ether under nitrogen and the reaction mixture was cooled to −78°C. n-butyllithium (3.2 mL, 8.06 mmol) was added dropwise. The reactionmixture was then stirred until the temperature was raised gradually to−30° C. 37 (1 g, 4.03 mmol) dissolved in 25 mL of dry ether was addeddropwise and the reaction mixture was allowed to stir until thetemperature warmed up to room temperature. 30 mL of water was added andthe organic phase was separated. The aqueous layer was then extractedwith ether (2×50), and the combined organic phases were dried overanhydrous magnesium sulfate, filtered and evaporated under reducedpressure. Purification of the crude product by column chromatography(silica gel, 30:70 EtOAc:Hexanes) yielded 1.1 g (66%) of 40 as whitecrystals. Rf: 0.15 (70:30, Hex:EA).

4-Isopropoxy-3-methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocycloheptene(41)

A mixture of the SM (40) (1.1 g, 2.64 mmol) in 30 mL of acetic acid and100 mL of water, taken in a 250 mL round bottom flask were refluxed for12 h. The reaction mixture was cooled and extracted with dichloromethane(3×25 mL) and the combined organic phases were dried over magnesiumsulfate. The solvent evaporated and the crude product was purified byflash column chromatography (10:90 ethylacetate:hexanes), to afford 1 g(95%) of4-Isopropoxy-3-methoxy-8-(3,4,5-trimethoxy-phenyl)-6,7-dihydro-5H-benzocyclohepteneas white crystals. Rf:56 (60:40, Hex:EA)

¹H NMR (CDCl₃, 300 MHz): δ 6.93 (d, J=8.46 Hz, 1H), δ 6.74 (d, J=8.49Hz, 1H), δ 6.71 (s, 1H), δ 6.70 (s, 2H), δ 4.40 (septet, J=6.18 Hz, 1H),δ 3.90 (s, 6H), δ 3.87 (s, 3H), δ 3.84 (s, 3H), δ 2.91 (t, J=6.06 Hz, 2H), δ 2.60 (t, J=6.78 Hz, 2H), δ 2.16 (p, J=6.54 Hz, 2H), δ 1.27 (d,J=6.18 Hz, 6H).

2-Methoxy-6-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H-benzocyclohepten-1-ol(3)

41 (220 mg, 0.55 mmol) was dissolved in 15 mL of anhydrousdichlromethane under nitrogen at room temperature. Aluminium chloride(147 mg, 1.1 mmol) was added and the reaction mixture was stirred for 10minutes. 10 mL of water was added and the product was extracted indichloromethane (2×20 mL) and the combined organic phases were driedover anhydrous sodium sulfate, evaporated under reduced pressurepurified by column chromatography. The product 3, 81 mg (41% yield) wasobtained in 10% ethylacetate in hexanes. Rf. 0.54 (60:40 Hex:EA).

¹H NMR (CDCl₃, 300 MHz): δ 6.76 (d, J=8.37 Hz, 1H), δ 6.71 (d, J=8.31Hz, 1 H), δ 6.70 (s, 3H), δ 5.71 (s, 1H), δ 3.91 (s, 3H), δ 3.90 (s,3H), δ 3.87 (s, 3H), δ 2.92 (t, J=6.12 Hz, 2H), δ 2.61 (t, J=6.12 Hz,2H), δ 2.19 (p, J=6.60 Hz, 2H). ¹³C NMR (CDCl₃, 75 MHz): δ 152.89,145.03, 142.49, 141.26, 140.24, 137.26, 131.69, 128.13, 126.83, 121.75,107.68, 103.52, 60.85, 56.11, 32.85, 29.80, 24.89

Example 2 Synthesis of Optional Linkers

The compounds of the invention may further comprise a benzoylsubstituent in which a carbonyl group is introduced between the coresuberene (or dihydronapthalene) ring and the pendant aryl ring.Furthermore, the carbonyl group of the benzoyl substituent can bereplaced with an oxygen to generate a new compound which maintains thesame or similar biological efficacy with tubulin. These compounds may beprepared by an addition elimination reaction utilizing thetrimethoxyphenolic anion as a nucleophile. Other linkage atoms betweenthe aryl-aryl rings are conceivable as well, including thioethers (—S—),secondary alcohols (—CH(OH)—, and methylenes (—CH2-). These compoundsare intended to form a one-atom bridge between the substituted aryl andthe chromene ring. For example, the secondary alcohols can be created byreduction of corresponding ketones (—C═O)— with sodium borohydride, andmethylenes can be created by reduction with trifluoroacetic acid.Alternatively, a single covalent bond can substitute for the 1-atomlinker.

Synthesis of(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(23; see FIG. 9)

(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(23) was prepared using methods known in the art (Atmaram et al, Bioorg.Med. Chem. Lett. (1999), 9, 2119; Koo, JACS. (1953), 75 (18), 1891;Pettit et al., J. Med. Chem. (2000), 43, 2731; Uffe et al., Tet Lett.(2005), 46, 4261) and as outlined below.

5-Oxo-5-(2,3,4-trimethoxy-phenyl)-pentanoic acid methyl ester (60)

75 g of polyphosphoric acid (Acros) was charged in a 250 mL round bottomflask, followed by addition of 1,2,3-trimethoxybenzene (5.0 g, 29.73mmol), and mono-methylglutarate (6.516 g, 44.60 mmol). The reactionmixture was stirred mechanically for 2.5 h at 45° C. The reactionmixture was then poured into a 1000 mL beaker containing around 250 mLof ice, and stirred well until all the product precipitated out. The tancolored product was then filtered and washed with water and dried undervacuum. No, purification required at this stage. 6.78 g (77%) of productwas obtained, which was pure by NMR.

¹H NMR (CDCl, 300 MHz): δ 7.48 (d, J=8.86 Hz, 1H), δ 6.71 (d, J=8.98 Hz,1 H), δ 3.96 (s, 3H), δ 3.91 (s, 3H), δ 3.87 (s, 3H), δ 3.67 (s, 3H), δ3.02 (t, J=7.14, 2H), δ 2.41 (t, J=7.40 Hz, 2H), δ 2.03 (p, J=7.16 Hz,2H).

5-Oxo-5-(2,3,4-trimethoxy-phenyl)-pentanoic acid (61)

6.78 g of sodium hydroxide was dissolved in 25 mL of methanol in a 100mL round bottom flask. The reaction mixture was cooled to roomtemperature and 60 (6.78 g, 22.90 mmol) was added followed by 5 mL ofwater and the reaction mixture was refluxed for 30 min. The solvent wasevaporated under reduced pressure and the reaction mixture wasneutralized with dilute hydrochloric acid and the reaction mixture wasextracted with ether (100×3) and the combined organic phases were washedwith water and dried over sodium sulfate and solvent evaporated underreduced pressure. The crude product was purified by columnchromatography (20:80 EA:Hex) to yield 7.05 g of 61 (quantitativeyield).

¹H NMR (CDCl₃, 300 MHz): δ 7.50 (d, J=8.88 Hz, 1H), δ 6.70 (d, J=8.91Hz, 1 H), δ 3.96 (s, 3H), δ 3.91 (s, 3H), δ 3.87 (s, 3H), δ 3.05 (t,J=7.08, 2H), δ 2.50 (t, J=7.68 Hz, 2H), δ 2.04 (p, J=6.95 Hz, 2H).

5-(2,3,4-Trimethoxy-phenyl)-pentanoic acid (62)

5-Oxo-5-(2,3,4-trimethoxy-phenyl)-pentanoic acid (7.05 g 24.97 mmol) wasdissolved in 100 mL of anhydrous ethanol under inert atmosphere and 2.0g of Pd—C was added. The nitrogen gas was removed by vacuum, andhydrogen gas was passed into the flask. The reaction mixture was stirredfor 1 h. The completion of the reaction was confirmed by TLC. Thereaction mixture was filtered through celite. The solvent was evaporatedunder reduced pressure to obtain 6.48 g (97.74%) of 62 (colorless oil)which was pure by NMR.

¹H NMR (CDCl₃, 300 MHz): δ 6.81 (d, J=8.48 Hz, 1H), δ 6.60 (d, J=8.50Hz, 1 H), δ 3.87 (s, 3H), δ 3.86 (s, 3H), δ 3.84 (s, 3H), δ 2.60 (t,J=7.65, 2H), δ 2.39 (t, J=7.42 Hz, 2H), δ 1.66 (m, 4H).

1,2,3-Trimethoxy-6,7,8,9-tetrahydro-benzocyclohepten-5-one (63)

60 (6.48 g, 24.19 mmol) was weighed in a 250 mL round bottom flask,followed by addition of 75 g of polyphosphoricacid. The reaction mixturewas stirred mechanically for 2.5 h at 45° C. The reaction mixture waspoured into 250 mL of ice and stirred until the entire polyphoshoricacid dissolved. The resultant solution was extracted withdichloromethane (100×3), and the combined organic phases were dried overanhydrous Na₂SO₄, filtered and evaporated under reduced pressure.Purification by column chromatography (92:8 Hexanes:Ethylacetate),yielded 3.0 g (50%) of 63. The product was pure by NMR.

¹H NMR (CDCl₃, 300 MHz): δ 7.13 (s, 1H), δ 3.93 (s, 3H), δ 3.88 (s, 3H),δ 3.84 (s, 3H), δ 2.94 (t, 2H), δ 2.73 (m, 2H), δ 1.81 (m, 4H)

¹³C NMR (CDCl₃, 75 MHz): δ 204.93, 151.53, 150.95, 145.86, 134.37,128.86, 107.44, 61.37, 60.84, 55.95, 40.75, 24.98, 22.93, 20.91.

(1,2,3-trimethoxy-6,7,8,9-tetrahydrobenzoheptene)-5-p-toluenesulfonylhydrazone(64)

(1,2,3-trimethoxy-6,7,8,9-tetrahydrobenzoheptene)-5-p-toluenesulfonylhydrazone(64) was prepared according to the methods of Pinney et al. (Pinney etal., Steroids. (1992), 57 (5), 222) and as outlined below.

64 (8.21 g, 32.81 mmol) was dissolved in 150 mL of absolute ethanolfollowed by p-toluenesulfonylhydrazide (6.11 g. 32.81 mmol) undernitrogen. The reaction mixture was stirred for five minutes at roomtemperature until the solid dissolved. P-toluenesulfonic acidmonohydrate (0.28 g, 0.05 mmol) was added and the reaction mixture wasallowed to stir for 12 h. The product precipitated out as white solid,which was then filtered and washed with ice-cold ethanol and dried(13.50 g, 98% yield). Rf: 0.325 (60:40 Hex:EA).

¹H NMR (DMSO-d6, 300 MHz): δ 10.33 (s, 1H), δ 7.83 (s-broad, 2H), δ 7.51(s-broad, 2H), δ 6.38 (s, 1H), δ 3.73 (s, 3H), δ 3.69 (s, 6H), δ 2.73(m, 7H), δ 1.60 (s, 2H), δ 1.47 (s, 2H).

(2,3-Bis-benzyloxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanol(94)

36 mL of freshly distilled TMEDA was charged in a 250 mL round bottomflask under nitrogen. n-BuLi (5.89 mL, 14.72 mmol) was added and thereaction mixture was cooled to −50° C. 64 (1.54 g, 3.68 mmol) was addedand the reaction mixture was then stirred to warm up to roomtemperature, which took approximately about 7 h.2,3-dibenzlyoxy-4-methoxybenzaldehyde (5.13 g, 14.72 mmol) was thenadded and the reaction mixture was stirred for 1 h. 25 mL of water wasadded and the product was extracted with ethyl acetate (2×100 mL). Thecombined organic phases were washed with aqueous CuSO₄, followed bybrine, dried over anhydrous sodium sulfate, filtered and evaporatedunder reduced pressure. Purification of the crude by columnchromatography (16:84, EA:Hex) yielded 1.02 g (48% yield) of product(94) as pale yellow oil. Rf: 0.56 (60:40 Hex:EA).

¹H NMR (CDCl₃, 300 MHz): δ 7.35 (m, 10H), δ 7.11 (d, J=8.66 Hz, 1H), δ6.67 (d, J=8.68 Hz, 1H), δ 6.63 (s, 1H), δ 6.06 (t, J=7.11 Hz, 1H), δ5.85 (d, J=4.27 Hz, 1H), δ 5.13 (d, J=10.90 Hz, 1H), δ 5.03 (d, J=10.90Hz, 1H), δ 5.00 (s, 2H), δ 3.85 (s, 3H), δ 3.82 (s, 6H), δ 3.63 (s, 3H),δ 2.50 (t, J=7.07, 2H), δ 2.04 (t, J=6.97, 2H), δ 1.84 (m, 2H).

(2,3-Bis-benzyloxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(95)

94 (0.94 g, 1.62 mmol) was dissolved in 20 mL of dry dichloromethane andwas added to a solution of Dess-Martin periodinane (2.7 g, 6.5 mmol) indry dichloromethane under nitrogen. The reaction mixture was stirred for1.30 h and then water (30 mL) was added, followed by 1.3 M NaOH solution(50 mL) and 50 mL of dichloromethane. The organic layer was washed with1.3 M NaOH solution (50 mL), followed by 100 mL of water. The organicphase was dried over anhydrous sodium sulfate, filtered and then thesolvent was removed under reduced pressure. The crude was purified bycolumn chromatography (10:90, EtOAc:Hexanes) to yield a pale yellow oil(0.69 g, 73% yield) as product. Rf: 0.43 (60:40 Hex:EA).

NMR data: ¹H(CDCl₃, 300 MHz): δ 7.40 (m, 10H), δ 7.11 (d, J=8.47 Hz,1H), δ 6.78 (t, J=7.30 Hz, 1H), δ 6.72 (d, J=8.54 Hz, 1H), δ 6.61 (s,1H), δ 5.09 (s, 2H), δ 5.02 (s, 2H), δ 3.90 (s, 3H), δ 3.89 (s, 3H), δ3.85 (s, 3H), δ 3.70 (s, 3H), δ 2.51 (t, J=6.50, 3H), δ 2.04 (m, 4H).

(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(23)

(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(23) was prepared according to the methods of Felix et al. (Felix, etal., J. Org. Chem. (1978), 43 (21), 4194) and as outlined below.

95 (0.69 g, 1.19 mmol) was dissolved in 20 mL of anhydrous EtOH undernitrogen and the reaction mixture was immersed in a water bath tomaintain a temperature of 25° C. 1.2 g of Pd—C was added followed by1,4-cyclohexadiene (1.13 mL, 11.90 mmol) and the reaction mixture wasstirred for 7 h. The reaction mixture was monitored for completion byTLC (Rf: 0.32, 60:40 Hex:EA). The reaction mixture was filtered throughcelite, washed with ethylacetate, rotavaped and purified by columnchromatography (20:80 EtOAc:Hexanes) to obtain 0.3 g (63%) of 23 asyellow oil.

NMR data: ¹H (CDCl₃, 300 MHz): δ 12.49 (s, 1H), δ 7.05 (d, J=9.03 Hz,1H), δ 6.66 (t, J=6.84 Hz, 1H), δ 6.41 (s, 1H), δ 6.37 (d, J=9.06 Hz,1H), δ 5.69 (s, 1H), δ 3.90 (s, 3H), δ 3.88 (2s, 6H), δ 3.69 (s, 3H), δ2.73 (t, J=6.52, 2H), δ 2.16 (m, 4H).

¹³C (CDCl₃, 75 MHz): δ 200.76, 151.99, 151.37, 151.07, 141.81, 141.45,139.10, 133.325, 132.38, 127.24, 125.16, 115.99, 114.33, 107.84, 102.54,61.55, 60.79, 56.11, 55.98, 33.87, 26.00, 23.82.

Synthesis of(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(24; see FIG. 9)(3-Isopropoxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanol(96)

20 mL of freshly distilled TMEDA was charged in a 250 mL round bottomflask under nitrogen. n-BuLi (4.8 mL, 9.60 mmol) was added and thereaction mixture was cooled to −50° C. 68 (1.0 g, 2.40 mmol) was addedand the reaction mixture was then stirred to warm up to roomtemperature, which took approximately about 7 h.3-isopropoxy-4-methoxybenzaldehyde (1.9 g, 9.60 mmol) was then added andthe reaction mixture was stirred for 1 h. 25 mL of water was added andthe product was extracted with ethyl acetate (2×50 mL). The combinedorganic phases were washed with aqueous CuSO₄, followed by brine, driedover anhydrous sodium sulfate, filtered and evaporated under reducedpressure. Purification of the crude by column chromatography (16:84,EA:Hex) yielded 0.3 g (29% yield) of 96 as pale yellow oil. Rf: 0.30(60:40 Hex:EA).

¹H NMR (CDCl₆, 300 MHz): δ 6.91 (m, 2H), δ 6.79 (d, J=8.22 Hz, 1H), δ6.65 (s, 1H), δ 6.27 (t, J=8.00 Hz, 1H), δ 5.53 (s, 1H), δ 4.43 (sep,J=6.09 Hz, 1H), δ 3.85 (s, 3H), δ 3.80 (2s, 6H), δ 3.71 (s, 3H), δ 2.42(m, 2H), δ 2.03 (m, 2H), δ 1.87 (m, 2H), δ 1.28 (d, J=6.02 Hz, 6H).

(3-Isopropoxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(97)

96 (0.30 g, 0.70 mmol) dissolved in 20 mL of dry dichloromethane wasadded to a solution of Dess-Martin periodinane (1.48 g, 3.50 mmol) indry dichloromethane under nitrogen. The reaction mixture was stirred for1.30 h at room temperature and then water (30 mL) was added, followed by1.3 M NaOH solution (50 mL) and 50 mL of dichloromethane. The organiclayer was washed with 1.3 M NaOH solution (50 mL), followed by 100 mL ofwater. The organic phase was dried over anhydrous sodium sulfate,filtered and then the solvent was removed under reduced pressure. Thecrude was purified by column chromatography (10:90, EtOAc:Hexanes) toyield a pale yellow oil (0.170 g, 57% yield) as product 97. Rf: 0.62(40:60 Hex:EA).

¹H NMR (CDCl₃, 300 MHz): δ 7.40 (dd, J=8.40 Hz, 2.03 Hz, H), δ 7.31 (d,J=2.00 Hz, 1H), δ 6.80 (m, 2H), δ 6.47 (s, 1H), δ 4.47 (sep, J=6.12 Hz,1H), δ 3.90 (s, 3H), δ 3.88 (2s, 6H), δ 3.69 (s, 3H), δ 2.75 (t, J=6.68Hz, 2H), δ 2.15 (m, 4H), δ 1.31 (d, J=6.07 Hz, 6H).

(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone(24)

97 (0.07 g, 0.16 mmol) was dissolved in 10 mL of anhydrous CH₂Cl₂ undernitrogen at 0° C. Anhydrous AlCl₃ (44 mg, 0.33 mmol) was added and thereaction mixture was stirred for 2 h at 0° C. Saturated NH₄Cl (5 mL) wasadded and the organic phase was separated. The aqueous layer wasextracted with CH₂Cl₂ (10 mL) and the combined organic phases were driedover Na₂SO₄, filtered, solvents evaporated under reduced pressure. Thecrude product was purified by column chromatography (30:70EtOAc:Hexanes) to yield tan colored oil as product 24 (35 mg, 56%yield). Rf: 0.23 (60:40 Hex:EA).

¹H NMR (CDCl₃, 300 MHz): δ 7.41 (d, J=2.06 Hz, 1H), δ 7.34 (dd, J=8.36Hz, 2.08 Hz, H), δ 6.84 (d, J=8.41 Hz, 1H), δ 6.79 (t, J=6.95 Hz, 1H), δ6.53 (s, 1H), δ 3.96 (s, 3H), δ 3.90 (s, 3H), δ 3.88 (s, 3H), δ 3.72 (s,3H), δ 2.72 (t, J=6.62 Hz, 2H), δ 2.14 (m, 4H).

¹³C NMR (CDCl₃, 75 MHz): δ 195.77, 151.19, 150.15, 145.16, 142.23,141.62, 141.21, 132.50, 131.65, 127.34, 132.43, 115.90, 109.67, 108.46,61.53, 60.80, 56.02,55.93, 33.82, 26.04, 23.75.

Synthesis of(3-Hydroxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanone(22; see FIG. 8) 4-Oxo-4-(2,3,4-trimethoxy-phenyl)-butyric acid methylester (55)

A mixture of 75 g of polyphosphoric acid (Acros),1,2,3-trimethoxybenzene (5.0 g, 29.73 mmol), and mono-methyl succinate(5.9 g, 44.59 mmol) were mechanically stirred at 45° C. for 2.5 h. Thereaction mixture was then poured into a 1000 mL beaker containing round250 mL of ice, and stirred well until all the product was precipitatedout. The tan colored product was then filtered and washed with water anddried under vacuum. 6.1 g (72%) of 55 was obtained, which was pure byNMR.

¹H NMR (CDCl₃, 300 MHz): δ 7.56 (d, J=8.89 Hz, 1H), δ 6.72 (d, J=8.91Hz, 1 H), δ 3.99 (s, 3H), δ 3.91 (s, 3H), δ 3.87 (s, 3H), δ 3.70 (s,3H), δ 3.31 (t, J=6.57, 2H), δ 2.71 (t, J=6.57 Hz, 2H).

4-Oxo-4-(2,3,4-trimethoxy-phenyl)-butyric acid (56)

5.95 g of sodium hydroxide was dissolved in 200 mL of methanol in a 500mL round bottom flask. The reaction mixture was cooled to roomtemperature and 55 (6.78 g, 22.90 mmol) was added followed by 20 mL ofwater and 100 mL of methanol and the reaction mixture was refluxed for30 min. The solvent was evaporated under reduced pressure and thereaction mixture was neutralized with dilute hydrochloric acid and thereaction mixture was extracted with ether (100×3) and the combinedorganic phases were washed with water and dried over sodium sulfate andsolvent evaporated under reduced pressure. The crude product waspurified by column chromatography (20:80 EtOAc:Hexanes) to yield 5.43 gof 56 as tan colored solid (94%).

¹H NMR (CDCl₃, 300 MHz): δ 7.55 (d, J=8.87 Hz, 1H), δ 6.72 (d, J=8.90Hz, 1 H), δ 3.99 (s, 3H), δ 3.91 (s, 3H), δ 3.87 (s, 3H), δ 3.33 (t,J=6.60, 2H), δ 2.74 (t, J=6.50 Hz, 2H).

4-(2,3,4-Trimethoxy-phenyl)-butyric acid (57)

56 (5.43 g 20.24 mmol) was charged in a 250 mL round bottom flask, and100 mL of anhydrous ethanol was added followed by addition of 1.0 g ofPd—C, and the reaction flask was put under vacuum, until all the air inthe reaction flask was evacuated. Then, hydrogen gas was passed into theflask using a balloon filled with hydrogen gas. The reaction mixture wasstirred for 1 h. The completion of the reaction was confirmed by TLC.The reaction mixture was filtered through celite. The solvent wasevaporated under reduced pressure to obtain 5.42 g (quantitative) of 57as colorless oil Rf. 0.45 (70:30 Hex:EA).

¹H NMR (CDCl₃, 300 MHz): δ 6.82 (d, J=8.40 Hz, 1H), δ 6.60 (d, J=8.47Hz, 1 H), δ 3.87(2s, 6H), δ 3.84 (s, 3H), δ 2.59 (t, J=7.19, 2H), δ 2.33(t, J=7.36 Hz, 2H), δ 1.89 (p, J=7.41 Hz, 2H).

5,6,7-Trimethoxy-3,4-dihydro-2H-naphthalen-1-one (58)

59 (4.44 g, 17.46 mmol) was charged in a 250 mL round bottom flask,followed by addition of 80 g of polyphosphoricacid. The reaction wasstirred mechanically for 4 h at 70° C. The reaction mixture was pouredinto 250 mL of ice and the product precipitated out as tan coloredsolid, which was filtered and dried under high vacuum to yield 2.96 g ofproduct (72%). Rf: 0.36 (70:30 Hex:EA)

¹H NMR (CDCl₃, 300 MHz): δ 7.39 (s, 1H), δ 3.94 (s, 3H), δ 3.89 (s, 3H),δ 3.86 (s, 3H) δ 2.88 (t, J=5.92, 2H), δ 2.60 (t, J=5.97 Hz, 2H), δ 2.08(p, J=6.10 Hz, 2H).

(5,6,7-trimethoxy-3,4-dihydro-2H-napthalene)-1-p-toluenesulfonylhydrazone(59)

(5,6,7-trimethoxy-3,4-dihydro-2H-napthalene)-1-p-toluenesulfonylhydrazone(59) was prepared according to the methods of Pinney et al. (Pinney etal., Steroids. (1992), 57 (5), 222) and as outlined below.

63 (2.96 g g, 12.53 mmol) was dissolved in 150 mL of absolute ethanolfollowed by p-toluenesulfonylhydrazide (2.33 g. 12.53 mmol) undernitrogen. The reaction mixture was stirred for five minutes at roomtemperature until the solid dissolved. p-Toluenesulfonic acidmonohydrate (0.11 g, 0.63 mmol) was added and the reaction mixture wasallowed to stir for 12 h. The product precipitated out as white solid,which was then filtered and washed with ice-cold ethanol and dried (4.65g, 92% yield).

(3-Isopropoxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanol(92)

25 mL of freshly distilled TMEDA was charged in a 100 mL round bottomflask under nitrogen. n-BuLi (4.65 mL, 9.89 mmol) was added and thereaction mixture was cooled to −50° C. 59 (1.0 g, 2.47 mmol) was addedand the reaction mixture was then stirred to warm up to roomtemperature, which took approximately about 7 h.3-isopropoxy-4-methoxybenzaldehyde (1.92 g, 9.89 mmol) was then addedand the reaction mixture was stirred for 1 h. 25 mL of water was addedand the product was extracted with ethyl acetate (2×100 mL). Thecombined organic phases were washed with aqueous CuSO₄, followed bybrine, dried over anhydrous sodium sulfate, filtered and evaporatedunder reduced pressure. Purification of the crude by columnchromatography (16:84, EA:Hex) yielded 0.70 g (69% yield) of product aspale yellow oil. Rf: 0.31 (40:60 EtOAc:Hexanes).

NMR data: ¹H (CDCl₃, 300 MHz): δ 6.98 (m, 2H), δ 6.66 (s, 1H), δ 6.16(t, J=4.57 Hz, 1H), δ 5.64 (s, 1H), δ 4.49 (sep, J=6.09 Hz, 1H), δ 3.84(s, 6H), δ 3.81 (s, 3H), δ 3.66 (s, 3H), δ 2.73 (t, J=7.80 Hz, 2H), δ2.31 (m, 2H), δ 1.31 (dd, J=6.09 Hz, 6H).

(3-Isopropoxy-4-methoxy-phenyl)-(5,6,7-trimethoxy-3,4-dihydro-naphthalen-1-yl)-methanone(93)

SM (92) (0.94 g, 1.62 mmol) was dissolved in 20 mL of drydichloromethane and was added to a solution of Dess-Martin periodinane(2.7 g, 6.5 mmol) in dry dichloromethane under nitrogen. The reactionmixture was stirred for 1.30 h and then water (30 mL) was added,followed by 1.3 M NaOH solution (50 mL) and 50 mL of dichloromethane.The organic layer was washed with 1.3 M NaOH solution (50 mL), followedby 100 mL of water. The organic phase was dried over anhydrous sodiumsulfate, filtered and then the solvent was removed under reducedpressure. The crude was purified by column chromatography (10:90,EtOAc:Hexanes) to yield a pale yellow oil (0.69 g, 73% yield) asproduct. Rf: 0.43 (40:60 EtOAc:Hexanes).

NMR data: ¹H (CDCl₃, 300 MHz): δ 7.49 (m, 2H), δ 6.87 (d, J=8.30 Hz,1H), δ 6.69 (s, 1H), δ 6.40 (t, J=4.70 Hz, 1H), δ 4.61 (sep, J=6.08 Hz,1H), δ 3.92 (s, 3H), δ 3.88 (s, 3H), δ 3.87 (s, 3H), δ 3.73 (s, 3H), δ2.84 (t, J=7.85 Hz, 2H), δ 2.44 (m, 2H), δ 1.37 (dd, J=6.09 Hz, 6H).

Synthesis of(7,8,9-trimethoxy-3,4,5,6-tetrahydro-2H-napthalene)-1-p-toluenesulfonylhydrazone(69; see FIG. 4) 6-Oxo-6-(2,3,4-trimethoxy-phenyl)-hexanoic acid methylester (65)

150 g of polyphosphoric acid (Acros) was added to a mixture of1,2,3-trimethoxybenzene (10.0 g, 59.47 mmol), and mono-methyl adipate(13.04 mL g, 89.20 mmol) in a 250 mL round bottom flask equipped with amechanical stirrer. The reaction mixture was stirred for 2.5 h at 45° C.The reaction mixture was then poured into a 1000 mL beaker containingaround 500 mL of ice, and stirred well. Tan colored product precipitatedout which was then filtered and washed with water and dried undervacuum. 17.18 g (93%) of 65 was obtained, which was pure by NMR. Rf:0.42 (40:60 EtOAc:Hexanes)

¹H NMR (CDCl₃, 300 MHz): δ 7.44 (d, J=8.84 Hz, 1H), δ 6.68 (d, J=8.88Hz, 1 H), δ 3.94 (s, 3H), δ 3.88 (s, 3H), δ 3.85 (s, 3H), δ 3.64 (s,3H), δ 2.95 (t, J=6.96, 2H), δ 2.34 (t, J=7.02 Hz, 2H), δ 1.68 (m, 4H).

6-Oxo-6-(2,3,4-trimethoxy-phenyl)-hexanoic acid (66)

17 g of sodium hydroxide was dissolved in 200 mL of methanol in a 1000mL round bottom flask. The reaction mixture was cooled to roomtemperature and 66 (6.78 g, 22.90 mmol) was added followed by 60 mL ofwater and 300 mL of anhydrous methanol, and the reaction mixture wasrefluxed for 30 min. The solvent was evaporated under reduced pressureand the reaction mixture was neutralized with dilute hydrochloric acidand the reaction mixture was extracted with ether (200×3) and thecombined organic phases were washed with water and dried over sodiumsulfate and solvent evaporated under reduced pressure. The crude productwas purified by column chromatography (30:70 EA:Hex) to yield 12.86 g of57 as pale yellow colored oil (78%). Rf: 0.15 (40:60 EtOAc:Hexanes).

¹H NMR (CDCl₃, 300 MHz): δ 7.47 (d, J=8.85 Hz, 1H), δ 6.70 (d, J=8.89Hz, 1 H), δ 3.96 (s, 3H), δ 3.90 (s, 3H), δ 3.87 (s, 3H), δ 2.98 (t,J=6.78, 2H), δ 2.41 (t, J=7.07 Hz, 2H), δ 1.72 (m, 4H).

6-(2,3,4-Trimethoxy-phenyl)-hexanoic acid (67)

57 (12.86 g 43.40 mmol) was charged in a 250 mL round bottom flask, and300 mL of anhydrous ethanol was added followed by addition of 4.0 g of10% wt. Pd—C, and the reaction flask was put under vacuum, until all theair in the reaction flask was evacuated. Then, hydrogen gas was passedinto the flask using a balloon filled with hydrogen gas. The reactionmixture was stirred for 12 h. The completion of the reaction wasconfirmed by TLC. The reaction mixture was filtered through celite. Thesolvent was evaporated under reduced pressure to obtain 11.7 g (96%) of67 as colorless oil Rf: 0.49 (50:50 Hex:EA).

¹H NMR (CDCl₃, 300 MHz): δ 6.80 (d, J=8.48 Hz, 1H), δ 6.59 (d, J=8.47Hz, 1 H), δ 3.86 (s, 6H), δ 3.83 (s, 3H), δ 2.53 (t, J=7.46, 2H), δ 2.33(t, J=7.41 Hz, 2H), δ 1.64 (m, 4H) δ 1.37 (m, 2H).

1,2,3-Trimethoxy-7,8,9,10-tetrahydro-6H-benzocycloocten-5-one (68)

A mixture of 61 (2.0 g, 7.06 mmol) and 40 g of polyphosphoricacid werestirred mechanically for 4 h at 45° C. The reaction mixture was pouredinto 100 mL of ice and the resultant aqueous solution was extracted withDCM (2×100 mL). The combined organic phases were washed with water,followed by brine, dried over anhydrous Na₂SO₄, filtered and evaporated.The crude product was then purified by column chromatography (7:93EtOAc:Hexanes), to obtain 1.32 g of 65 as colorless oil (71%). Rf: 0.38(75:25 Hexanes:EtOAc)

¹H NMR (CDCl₃, 300 MHz): δ 6.99 (s, 1H), δ 3.91 (s, 3H), δ 3.90 (s, 3H),δ 3.89 (s, 3H) δ 2.90 (t, J=5.95, 2H), δ 2.57 (t, J=6.86 Hz, 2H), δ 1.82(m, 2H), δ 1.52 (m, 2H), δ 1.27 (m, 2H).

(5,6,7-trimethoxy-3,4-dihydro-2H-napthalene)-1-p-toluenesulfonylhydrazone(69)

68 (1.32 g g, 4.99 mmol) was dissolved in 30 mL of absolute ethanolfollowed by p-toluenesulfonylhydrazide (0.93 g. 4.99 mmol) undernitrogen. The reaction mixture was stirred for five minutes at roomtemperature until the solid dissolved. p-Toluenesulfonic acidmonohydrate (0.043 g, 0.25 mmol) was added and the reaction mixture wasallowed to stir for 12 h. The product precipitated out as white solid,which was then filtered and washed with ice-cold ethanol and dried (1.27g, 59% yield).

¹H NMR (DMSO-d₆, 300 MHz): δ 10.60 (s, 1H), δ 7.72 (d, J=8.08 Hz, 2H), δ7.36 (d, J=8.18 Hz, 2H), δ 6.41 (s, 1H), δ 3.73 (s, 6H), δ 3.41 (s, 3H),δ 3.33 (s, 3H) δ 2.55 (m, 4H), δ 1.23 (m, 4H), δ 1.03 (m, 4H).

FIG. 5 (lower panel) depicts a route for the synthesis of Compound 9.

FIG. 3 depicts a route for the synthesis of Compound 5.

FIG. 5 (upper panel) depicts a route for the synthesis of Compound 10.

Example 3 Synthesis of Intermediate I (FIG. 11)3-hydroxy,4-methoxybromobenzene

In a 500 mL round bottom flask was charged 2,4-dibromoanisole (5.0 g,18.8 mmol) followed by 200 mL of dry tetrahydrofuran under nitrogen. Thereaction mixture was then cooled to −78° C., and n-butyllithium (16.11mL, 22.56 mmol) was added dropwise. The reaction mixture was stirred for30 min at −78° C. The reaction mixture was then warmed to 0° C. andtrimethylborate (2.57 mL, 22.56 mmol) was added dropwise. The reactionmixture was stirred at room temperature for 30 min. 5 mL of glacialacetic acid was added followed by 10 mL of 35% wt. hydrogen peroxide ina drop wise fashion. The reaction was allowed to stir 12 h at roomtemperature. The reaction was quenched with 1 N HCl, extracted withethylacetate (100×3) and the combined organic phases were dried overNa₂SO₄, filtered and evaporated under reduced pressure. Purification bycolumn chromatography yielded 1 g (26%) of 3-hydroxy,4-methoxybromobenzene (white crystals). (96:4 Hexanes:ethylacetate).

Example 4 Inhibition of Tubulin Polymerization

IC₅₀ values for tubulin polymerization were determined according to apreviously described procedure (Bai et al., Cancer Research, 1996) andare summarized in Table 3 below. Purified tubulin is obtained frombovine brain cells as previously described (Hamel and Lin, Biochemistry,1984). Various amounts of inhibitor were preincubated for 15 minutes at37° C. with purified tubulin. After the incubation period, the reactionwas cooled and GTP was added to induce tubulin polymerization.Polymerization was then monitored in a Gilford spectrophotometer at 350nm. The final reaction mixtures (0.25 ml) contained 1.5 mg/ml tubulin,0.6 mg/ml microtubule-associated proteins (MAPs), 0.5 mM GTP, 0.5 mlMMgCl₂, 4% DMSO and 0.1M 4-morpholineethanesulfonate buffer (MES, pH6.4). IC50 is the amount of inhibitor needed to inhibit tubulinpolymerization 50% with respect to the amount of inhibition that occursin the absence of inhibitor.

TABLE 3 In vitro inhibition of tubulin polymerization: Compound IC50(μM) CA-4 0.73 1 ~40 2 1.4 ± 0.2 3 ~40 23 ~40 24 ~40

Example 5 In vitro Cytotoxicity Activity Against Cancer Cell Lines

Newly prepared compounds were evaluated for cytotoxic activity against avariety of cell lines derived from human tumors using an assay systemsimilar to the National Cancer Institute procedure previously described(Monks et al., J. Natl. Cancer Inst., 1991). Briefly, the cellsuspensions, diluted according to the particular cell type and theexpected target cell density (5,000-40,000 cells per well based on cellgrowth characteristics), were added by pipet (100 ul) to 96-wellmicrotiter plates. Inoculates were allowed a preincubation time of 24-28hours at 37 C for stabilization. Incubation with the inhibitor compoundslasted for 48 hours in 5% CO₂ atmosphere and 100% humidity.Determination of cell growth was performed by in situ fixation of cells,followed by staining with a protein-binding dye sulforhodamine B (SRB),which binds to the basic amino acids of cellular macromolecules. Thesolubilized stain was measured spectrophotometrically.

Several compounds were evaluated for cytotoxic activity against humanP388 leukemia cell lines. The effective dose or ED50 value (defined asthe effective dosage required for inhibition of 50% of cell growth) wasmeasured. These and additional compounds were evaluated in terms ofgrowth inhibitory activity against several other human cancer cell linesincluding: central nervous system (“CNS”, SF-268), pancreas (BXPC-3),non-small cell lung cancer (“lung-NSC”, NCI-H460), breast (MCF-7), colon(KM20L2), ovarian (OVCAR-3), and prostate (DU-145). The results aredescribed in Table 4 below. The growth inhibition GI50 (defined as thedosage required for inhibition of tumor cell growth by 50%) is listedfor each cell line.

TABLE 4 In vitro Cytotoxicity against Human Cancer Cell Lines ED50(μM/mL) for cell line GI50 (μM/mL) for Cell Line Compound P388 SF-268BXP-3 NCI-H460 MCF-7 KM20L2 DU-145 1 0.51 0.28 0.40 0.23 0.32 0.20 0.332 7.4 >10 >10 >10 >10 >10 >10 23 0.029 0.0090 0.017 0.023 0.0059 0.0310.026

Example 6 Inhibition of Tumor Blood Flow

The antivascular effects of the compounds of the invention are assessedin tumor-bearing mice using a Fluorescent Bead Assay. A MHEC-5Themangioendothelioma tumor model is established by subcutaneousinjection of 0.5×106 cultured transformed cell murine myocardialvascular endothelial cell line (“MHEC5-T”) cells into the right flank ofFox Chase CB-17 Severe Combined Immunodeficient (“SCID”) mice. Whentransplanted tumors reach a size of 500 mm³ (a size without developmentof necrosis), the mice receive a single intraperitoneal (i.p.) injectionof saline control or compound at doses ranging from 0.1 to 50 mg/kg. At24 hours post-treatment, mice are injected intravenously with 0.25 ml ofdiluted FluoSphere beads (1:6 in physiological saline) in the tail vein,sacrificed after 3 minutes, and tumor is excised for cryosectioning.Tumor cryosections at a thickness of 8 um is directly examined usingquantitative fluorescent microscopy. Blood vessels are indicated by bluefluorescence from injected beads. For quantification, image analysis of3 sections from three tumors treated in each group is examined andvascular shutdown is expressed as vessel area (mm²) per tumor tissuearea (mm²) as a percentage of the control (“% VAPM”).

Example 7 Evaluation of Tumor Growth Control in vivo by Hollow FiberAssay

Human tumor cells are grown in polyvinylidene fluoride (PVDF) hollowfibers and each cell line is injected into the mice intraperitoneal (IP)and subcutaneous (SC) membrane compartments. Mice are injectedintraperitoneally with two different test doses of compound. Controlanimals are injected with the diluent. A formazan dye (MTT) conversionassay is used to determine viable cell mass for the assessment of theanti-cancer effects of the ligand. The % T/C is calculated using averageoptical density of the compound treated sample divided by the averageoptical density of the control animals.

Alternative Embodiments

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

It should be readily apparent to any practitioner skilled in the artthat there are various ways of appending trimethoxyaryl andtrimethoxyaroyl groups around a Combrestatin analog molecular scaffoldin a manner which will result in a similar molecular conformationcapable of undergoing pseudo pi-pi stacking. In addition, although thetrimethoxyaryl motif seems optimal for enhanced tubulin binding, it isalso very possible that another combination of alkoxy substituents (suchas ethoxy, propoxy, isopropoxy, allyloxy, etc.) either as atrisubstituted pattern or as disubstituted (with one type of alkoxymoiety) and monosubstituted (with a different alkoxy moiety), or withthree distinct types of alkoxy moieties may also have good tubulinbinding characteristics. It is also conceivable that instead of havingaryl alkoxy groups, it may be possible to substitute simply aryl-alkyland aryl-alkenyl moieties and still maintain the enhanced cytotoxicityprofile. Phenolic groups may also have activity on these describedchromene ligands. The synthesis of any of these modifiedchromene-ligands will be very straight-forward for anyone skilled in theart, and often will only involve a different choice of initial startingmaterials. To prepare these alternative ligands, the same syntheticschemes as disclosed herein or similar schemes with only slightmodifications may be employed.

1. A compound of the Formula IV:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindependently indicate a single or double bond; X is C(O); R₁, R₂, R₃,R₄, R₅, R₆ and R₇ are each, independently, selected from the groupconsisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl, amine,phosphate, phosphoramidate, and amino acid acyl group; and phenyl ring“Z” is bonded to either carbon “a” or “b.”
 2. The compound of claim 1,wherein the compound of Formula IV is selected from the group consistingof (15)(1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-6-yl)-(3,4,5-trimethoxy-phenyl)-methanone;(16) (1-Hydroxy-2-methoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-(3,4,5trimethoxy-phenyl )-methanone; (23)(2,3-Dihydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7H-benzocyclohepten-5-yl)-methanone,and (24)(3-Hydroxy-4-methoxy-phenyl)-(1,2,3-trimethoxy-8,9-dihydro-7Hbenzocyclohepten-5-yl)-methanone.3. A compound of the Formula I:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindicate a single or double bond; R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, halogen,lower alkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate,and amino acid acyl group; X is C(O); and n is
 2. 4. The compound ofclaim 3, wherein the ring-substituted bicyclic fused ring system isrepresented by a compound of the Formula II:

or a pharmaceutically acceptable salt thereof, wherein the dashed linesindicate a single or double bond; R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areeach, independently, selected from the group consisting of H, halogen,lower alkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate,and amino acid acyl group; X is C(O); and n is
 2. 5. A compound of claim1 of the Formula IIa:

or a pharmaceutically acceptable salt thereof, wherein phenyl ring “Z”is bonded to either carbon “a” or “b”; R₁, R₃, R₄ and R₅ are each,independently, selected from the group consisting of H, lower alkoxy andhydroxyl; X is C(O); and n is
 3. 6. A compound of claim 1 of the FormulaIIb:

or a pharmaceutically acceptable salt thereof, wherein phenyl ring “Z”is bonded to either carbon “a” or “b”; R₁, R₃, R₄ and R₅ are each,independently, selected from the group consisting of H, lower alkoxy andhydroxyl; X is C(O); and n is
 3. 7. The compound of claim 1, wherein Zis bonded to carbon “a.”
 8. The compound of claim 1, wherein Z is bondedto carbon “b.”
 9. The compound of claim 1, wherein at least one of R₁-R₆is lower alkoxy.
 10. The compound of claim 1, wherein R₇ is H.
 11. Thecompound of claim 3, wherein at least one of R₁-R₆ is lower alkoxy. 12.The compound of claim 3, wherein R₇ is H.
 13. The compound of claim 5,wherein Z is bonded to carbon “a.”
 14. The compound of claim 6, whereinZ is bonded to carbon “a.”
 15. The compound of claim 6, wherein Z isbonded to carbon “b.”